Anti-angiogenic compositions and methods of use

ABSTRACT

The present invention provides compositions comprising an anti-angiogenic factor, and a polymeric carrier. Representative examples of anti-angiogenic factors include Anti-Invasive Factor, Retinoic acids and derivatives thereof, and paclitaxel. Also provided are methods for embolizing blood vessels, and eliminating biliary, urethral, esophageal, and tracheal/bronchial obstructions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of co-pending PCT applicationCA94/00373, filed Jul. 19, 1994. In addition, this application is acontinuation-in-part of U.S. patent application Ser. No. 08/094,536,filed Jul. 19, 1993.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions andmethods for treating cancer and other angiogenic-dependent diseases, andmore specifically, to compositions comprising anti-angiogenic factorsand polymeric carriers, stents which have been coated with suchcompositions, as well as method for utilizing these stents andcompositions.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis-dependent diseases (i.e., those diseases whichrequire or induce vascular growth) represent a significant portion ofall diseases for which medical treatment is sought. For example, canceris the second leading cause of death in the United States, and accountsfor over one-fifth of the total mortality. Briefly, cancer ischaracterized by the uncontrolled division of a population of cellswhich, most typically, leads to the formation of one or more tumors.Such tumors are also characterized by the ingrowth of vasculature whichprovide various factors that permit continued tumor growth. Althoughcancer is generally more readily diagnosed than in the past, many forms,even if detected early, are still incurable.

[0004] A variety of methods are presently utilized to treat cancer,including for example, various surgical procedures. If treated withsurgery alone however, many patients (particularly those with certaintypes of cancer, such as breast, brain, colon and hepatic cancer) willexperience recurrence of the cancer. Therefore, in addition to surgery,many cancers are also treated with a combination of therapies involvingcytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine,cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy. Onedifficulty with this approach, however, is that radiotherapeutic andchemotherapeutic agents are toxic to normal tissues, and often createlife-threatening side effects. In addition, these approaches often haveextremely high failure/remission rates.

[0005] In addition to surgical, chemo- and radiation therapies, othershave attempted to utilize an individual's own immune system in order toeliminate cancerous cells. For example, some have suggested the use ofbacterial or viral components as adjuvants in order to stimulate theimmune system to destroy tumor cells. (See generally “Principles ofCancer Biotherapy,” Oldham (ed.), Raven Press, New York, 1987.) Suchagents have generally been useful as adjuvants and as nonspecificstimulants in animal tumor models, but have not as of yet proved to begenerally effective in humans.

[0006] Lymphokines have also been utilized in the treatment of cancer.Briefly, lymphokines are secreted by a variety of cells, and generallyhave an effect on specific cells in the generation of an immuneresponse. Examples of lymphokines include Interleukins (IL)-1, -2, -3,and -4, as well as colony stimulating factors such as G-CSF, GM-CSF, andM-CSF Recently, one group has utilized IL-2 to stimulate peripheralblood cells in order to expand and produce large quantities of cellswhich are cytotoxic to tumor cells (Rosenberg et al., N. Engl. J. Med.313:1485-1492, 1985).

[0007] Others have suggested the use of antibodies in the treatment ofcancer. Briefly, antibodies may be developed which recognize certaincell surface antigens that are either unique, or more prevalent oncancer cells compared to normal cells. These antibodies, or “magicbullets,” may be utilized either alone or conjugated with a toxin inorder to specifically target and kill tumor cells (Dillman, “AntibodyTherapy,” Principles of Cancer Biotherapy, Oldham (ed.), Raven Press,Ltd.. New York, 1987). However, one difficulty is that most monoclonalantibodies are of murine origin, and thus hypersensitivity against themurine antibody may limit its efficacy, particularly after repeatedtherapies. Common side effects include fever, sweats and chills, skinrashes, arthritis, and nerve palsies.

[0008] One additional difficulty of present methods is that localrecurrence and local disease control remains a major challenge in thetreatment of malignancy. In particular, a total of 630,000 patientsannually (in the U.S.) have localized disease (no evidence of distantmetastatic spread) at the time of presentation, this represents 64% ofal those patients diagnosed with malignancy (this does not includenonmelanoma skin cancer or carcinoma in situ). For the vast majority ofthese patients, surgical resection of the disease represents thegreatest chance for a cure and indeed 428,000 will be cured after theinitial treatment—428,000. Unfortunately. 202,000 (or 32% of allpatients with localized disease) will relapse after the initialtreatment. Of those who relapse, the number who will relapse due tolocal recurrence of the disease amounts to 133,000 patients annually (or21% of all those with localized disease). The number who will relapsedue to distant metastases of the disease is 68,000 patients annually(11% of all those with localized disease). Another 102,139 patientsannually will die as a direct result of an inability to control thelocal growth of the disease.

[0009] Nowhere is this problem more evident than in breast cancer, whichaffects 186,000 women annually in the U.S. and whose mortality rate hasremained unchanged for 50 years. Surgical resection of the diseasethrough radical mastectomy, modified radical mastectomy, or lumpectomyremains the mainstay of treatment for this condition. Unfortunately, 39%of those treated with lumpectomy alone will develop a recurrence of thedisease, and surprisingly, so will 25% of those in which the resectionmargin is found to be clear of tumor histologically. As many as 90% ofthese local recurrences will occur within 2 cm of the previous excisionsite.

[0010] Similarly, in 1991, over 113,000 deaths and 238,600 new cases ofliver metastasis were reported in North America alone. The mean survivaltime for patients with liver metastases is only 6 6 months once liverlesions have developed. Non-surgical treatment for hepatic metastasesinclude systemic chemotherapy, radiation, chemoembolization, hepaticarterial chemotherapy, and intraarterial radiation. However, despiteevidence that such treatments can transiently decrease the size of thehepatic lesions (e.g., systemic chemotherapy and hepatic arterialchemotherapy initially reduces lesions in 15-20%, and 80% of patients,respectively), the lesions invariably reoccur. Surgical resection ofliver metastases represents the only possibility for a cure, but such aprocedure is possible in only 5%, of patients with metastases, and inonly 15-20% of patients with primary hepatic cancer.

[0011] One method that has been attempted for the treatment of tumorswith limited success is therapeutic embolization. Briefly, blood vesselswhich nourish a tumor are deliberately blocked by injection of anembolic material into the vessels. A variety of materials have beenattempted in this regard, including autologous substances such as fat,blood clot, and chopped muscle fragments, as well as artificialmaterials such as wool, cotton, steel balls, plastic or lass beads,tantalum powder, silicone compounds, radioactive particles, sterileabsorbable gelatin sponge (Sterispon, Gelfoam), oxidized cellulose(Oxycel), steel coils, alcohol, lyophilized human dura mater (Lyodura),microfibrillar collagen (Avitene), collagen fibrils (Tachotop),polyvinyl alcohol sponge (PVA; Ivalon), Barium-impregnated siliconspheres (Biss) and detachable balloons. The size of liver metastases maybe temporarily decreased utilizing such methods, but tumors typicallyrespond by causing the growth of new blood vessels into the tumor.

[0012] A related problem to tumor formation is the development ofcancerous blockages which inhibit the flow of material through bodypassageways, such as the bile ducts, trachea, esophagus, vasculature andurethra. One device, the stent, has been developed in order to hold openpassageways which have been blocked by tumors or other substances.Representative examples of common stents include the Wallstent, Streckerstent, Gianturco stent, and the Palmaz stent. The major problem withstents, however, is that they do not prevent the ingrowth of tumor orinflammatory material through the interstices of the stent. If thismaterial reaches the inside of a stent and compromises the stent lumen,it may result in blockage of the body passageway into which it has beeninserted. In addition, presence of a stent in the body may inducereactive or inflammatory tissue (e.g., blood vessels, fibroblasts, whiteblood cells) to enter the stent lumen, resulting in partial or completeclosure of the stent.

[0013] The present invention provides compositions and methods suitablefor treating cancers, as well as other non-tumorigenicangiogenesis-dependent diseases, and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

[0014] Briefly stated, the present invention provides anti-angiogeniccompositions, as well as methods and devices which utilize suchcompositions for the treatment of cancer and otherangiogenesis-dependent diseases. Within one aspect of the presentinvention, compositions are provided (anti-angiogenic compositions)comprising (a) an anti-angiogenic factor and (b) a polymeric carrier. Awide variety of molecules may be utilized within the scope of thepresent invention as anti-angiogenic factors, including for exampleAnti-Invasive Factor, retinoic acids and their derivatives, paclitaxelincluding analogues and derivatives thereof Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2.Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor-2,and lighter “d group” transition metals. Similarly, a wide variety ofpolymeric carriers may be utilized, representative examples of whichinclude poly (ethylene-vinyl acetate) (40% cross-linked), poly(D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomersand polymers, poly (glycolic acid), copolymers of lactic acid andglycolic acid, poly (caprolactone), poly (valerolactone), poly(anhydrides), copolymers of poly (caprolactone) or poly (lactic acid)with polyethylene glycol, and blends thereof.

[0015] Within certain preferred embodiments, the compositions comprise acompound which disrupts microtubule function, such as, for example,paclitaxel, estramustine, colchicine, methotrexate, curacin-A,epothilone, vinblastine or tBCEV. Within other preferred embodiments,the compositions comprise a polymeric carrier and a lighter d groputransition metal (e.g., a vanadium species, molybdenum species, tungstenspecies, titanium species, niobium species or tantalum species) whichinhibits the formation of new blood vessels.

[0016] Within one embodiment of the invention, the composition has anaverage size of 15 to 200 μm, within other embodiments, the polymericcarrier of the composition has a molecular weight ranging from less than1,000 daltons to greater than 200,000 to 300,000 daltons. Within yetother embodiments, the compositions provided herein may be formed intofilms with a thickness of betweem 100 μm and 2 mm, or thermologicallyactive compositions which are liquid at one temperature (e.g., above 45°C.) and solid or semi-solid at another (e.g., 37° C.).

[0017] Within another aspect of the present invention methods forembolizing a blood vessel are provided, comprising the step ofdelivering into the vessel a therapeutically effective amount of ananti-angiogenic composition (as described above), such that the bloodvessel is effectively occluded. Within one embodiment, theanti-angiogenic composition is delivered to a blood vessel whichnourishes a tumor.

[0018] Within vet another aspect of the present invention, stents areprovided comprising a generally tubular structure, the surface beingcoated with one or more anti-angiogenic compositions. Within otheraspects of the present invention, methods are provided for expanding thelumen of a body passageway, comprising inserting a stent into thepassageway, the stent having a generally tubular structure, the surfaceof the structure being coated with an anti-angiogenic composition asdescribed above, such that the passageway is expanded. Within variousembodiments of the invention, methods are provided for eliminatingbiliary obstructions, comprising inserting a biliary stent into abiliary passageway; for eliminating urethral obstructions, comprisinginserting a urethral stent into a urethra; for eliminating esophagealobstructions, comprising inserting an esophageal stent into anesophagus; and for eliminating tracheal/bronchial obstructions,comprising inserting a tracheal/bronchial stent into the trachea orbronchi. In each of these embodiments, the stent has a generally tubularstructure, the surface of which is coated with an anti-angiogeniccomposition as described above.

[0019] Within another aspect of the present invention, methods areprovided for treating tumor excision sites, comprising administering ananti-angiogenic composition as described above to the resection marginsof a tumor subsequent to excision, such that the local recurrence ofcancer and the formation of new blood vessels at the site is inhibited.Within yet another aspect of the invention, methods for treating cornealneovascularization are provided, comprising the step of administering toa patient a therapeutically effective amount of an anti-angiogeniccomposition as described above to the cornea, such that the formation ofblood vessels is inhibited. Within one embodiment, the anti-angiogeniccomposition further comprises a topical corticosteroid.

[0020] Within another aspect of the present invention, methods areprovided for inhibiting angiogenesis in patients with non-tumorigenic,angiogenesis-dependent diseases, comprising administering to a patient atherapeutically effective amount of paclitaxel to a patient with anon-tumorigenic angiogenesis-dependent disease, such that the formationof new blood vessels is inhibited. Within other aspects, methods areprovided for embolizing blood vessels in non-tumorigenic,angiogenesis-dependent diseases, comprising delivering to the vessel atherapeutically effective amount of a composition comprising paclitaxel,such that the blood vessel is effectively occluded.

[0021] Within yet other aspects of the present invention, methods areprovided for expanding the lumen of a body passageway, comprisinginserting a stent into the passageway, the stent having a generallytubular structure, the surface of the structure being coated with acomposition comprising paclitaxel, such that the passageway is expanded.Within various embodiments of the invention, methods are provided foreliminating biliary obstructions, comprising inserting a biliary stentinto a biliary passageway; for eliminating urethral obstructions,comprising inserting a urethral stent into a urethra; for eliminatingesophageal obstructions, comprising inserting an esophageal stent intoan esophagus; and for eliminating tracheal/bronchial obstructions,comprising inserting a trachea/bronchial stent into the trachea orbronchi. Within each of these embodiments the stent has a generallytubular structure, the surface of the structure being coated with acomposition comprising paclitaxel.

[0022] Within another aspect of the present invention, methods areprovided for treating a tumor excision site, comprising administering acomposition comprising paclitaxel to the resection margin of a tumorsubsequent to excision, such that the local recurrence of cancer and theformation of new blood vessels at the site is inhibited. Within otheraspects, methods are provided for treating neovascular diseases of theeye, comprising administering to a patient a therapeutically effectiveamount of an anti-angiogenic factor (such as a compound which disruptsmicrotubule function) to the eye, such that the formation of new vesselsis inhibited.

[0023] Within other aspects of the present invention, methods areprovided for treating inflammatory arthritis, comprising administeringto a patient a therapeutically effective amount of an anti-angiogenicfactor (such as a compound which disrupts microtubule function), or acomposition comprising an anti-angiogenic factor and a polymeric carrierto a joint. Within preferred embodiments, the anti-angiogenic factor maybe a compound which disrupts microtubule function such as paclitaxel, oran element from the lighter ‘d group’ transition metals, such as avanadium species.

[0024] Within yet another aspect of the invention, pharmaceuticalproducts are provided, comprising (a) a compound which disruptsmicrotubule function, in a container, and (b) a notice associated withthe container in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of a compound which disrupts microtubulefunction, for human or veterinary administration to treatnon-tumorigenic angiogenesis-dependent diseases such as, for example,inflammatory arthritis or neovascular diseases of the eve. Briefly,Federal Law requires that the use of a pharmaceutical agent in thetherapy of humans be approved by an agency of the Federal government.Responsibility for enforcement (in the United States) is with the Foodand Drug Administration, which issues appropriate regulations forsecuring such approval, detailed in 21 U.S C §§ 301-392. Regulation forbiological materials comprising products made from the tissues ofanimals, is also provided under 42 U.S.C. § 262. Similar approval isrequired by most countries, although, regulations may vary from countryto country.

[0025] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth belowwhich describe in more detail certain procedures, devices orcompositions, and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1A is a photograph which shows a shell-less ego culture onday 6. FIG. 1B is a digitized computer-displayed image taken with astereomicroscope of living, unstained capillaries (1040×). FIG. 1C is aphotograph of a corrosion casting which shows CAM microvasculature thatare fed by larger, underlying vessels (arrows; 1300×). FIG. 1D is aphotograph which depicts a 0.5 mm thick plastic section cut transverselythrough the CAM, and recorded at the light microscope level. Thisphotograph shows the composition of the CAM, including an outerdouble-layered ectoderm (Ec), a mesoderm (M) containing capillaries(arrows) and scattered adventitila cells, and a single layered endoderm(En) (400×). FIG. 1E is a photograph at the electron microscope level(3500×) wherein typical capillary structure is presented showingthin-wailed endothelial cells (arrowheads) and an associated pericyte.

[0027]FIGS. 2A, 2B, 2C and 2D are a series of digitized images of fourdifferent, unstained CAMs taken after a 48 hour exposure to digitizedimages of four different living, unstained CAMs were taken after a 48 hexposure to 10μ paclitaxel per ml of methylcellulose. The transparentmethylcellulose disk (*) containing paclitaxel is present on each CAMand is positioned over a singular avascular zone (A) with surroundingblood islands (Is). These avascular areas extend beyond the disk andtypically have a diameter of 6 mm. FIG. 2D illustrates the typical“elbowing” effect (arrowheads) of both small and large vessels beingredirected away from the periphery of the avascular zone.

[0028]FIG. 3A is a photograph (Mag=400×) which shows Just peripheral tothe avascular zone, that capillaries (arrowheads) exhibit numerousendothelial cells arrested in mitosis. Ectoderm (Ec), Mesoderm (M);Endoderm (En). FIG. 3B (Mag=400×) shows that within the avascular zoneproper the typical capillary structure has been eliminated and there arenumerous extravasated blood cells (arrowheads). FIG. 3C (Mag=400×) showsthat in the central area of the avascular zone, red blood cells aredispersed throughout the mesoderm.

[0029]FIGS. 3A, 3B and 3C are a series of photographs of 0.5 mm thickplastic sections transversely cut through a paclitaxel-treated CAM atthree different locations within the avascular zone.

[0030]FIGS. 4A, 4B and 4C are series of electron micrographs which weretaken from locations similar to that of FIGS. 3A, 3B and 3C(respectively) above.

[0031]FIG. 4A (Mag=2,200×) shows a small capillary lying subjacent tothe ectodermal layer (Ec) possessing three endothelial cells arrested inmitosis (*). Several other cell types in both the ectoderm and mesodermare also arrested in mitosis. FIG. 4B (Mag=2,800×) shows the earlyavascular phase contains extravasated blood cells subjacent to theectoderm; these blood cells are intermixed with presumptive endothelialcells (*) and their processes. Degrative cellular vacuoles (arrowhead).FIG. 4C (Mag=2,800×) shows that in response to paclitaxel, theecto-mesodermal interface has become populated with cells in variousstages of degradation containing dense vacuoles and granules(arrowheads)

[0032]FIG. 5 is a bar graph which depicts the size distribution ofmicrospheres by number (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

[0033]FIG. 6 is a bar graph which depicts the size distribution ofmicrospheres by weight (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

[0034]FIG. 7 is a graph which depicts the weight of encapsulation ofSodium Suramin in 50 mg poly (ethylene-vinyl acetate).

[0035]FIG. 8 is a graph which depicts the percent of encapsulation ofSodium Suramin in 50 mg poly (ethylene-vinyl acetate)

[0036]FIG. 9 is a bar graph which depicts the size distribution byweight of 5% ELVAX microspheres containing 10 mg sodium suramin made in5% PVA containing 10% NaCl.

[0037]FIG. 10 is a bar graph which depicts the size distribution byweight of 5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

[0038]FIG. 11 is a bar graph which depicts the size distribution bynumber of 5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

[0039]FIG. 12 is a line graph which depicts the time course of sodiumsuramin release.

[0040]FIG. 13 is an illustration of a representative embodiment ofhepatic tumor embolization.

[0041]FIG. 14 is an illustration of the insertion of a representativestent coated with an anti-angiogenic composition.

[0042]FIG. 15A is a graph which shows the effect of the EVA:PLA polymerblend ratio upon aggregation of microspheres. FIG. 15B is a scanningelectron micrograph which shows the size of “small” microspheres. FIG.15C (which includes a magnified inset—labelled “15C-inset”) is ascanning electron micrograph which shows the size of “large”microspheres. FIG. 15D is a graph which depicts the time course of invitro paclitaxel release from 0.6% w/v paclitaxel-loaded 50:50 EVA:PLApolymer blend microspheres into phosphate buffered saline (pH 7.4) at37° C. Open circles are “small” sized microspheres, and closed circlesare “large” sized microspheres. FIG. 15F is a photograph of a CAM whichshows the results of paclitaxel release by microspheres (“MS”). FIG. 15Fis a photograph similar to that of 15E at increased magnification.

[0043]FIG. 16 is a graph which shows release rate profiles frompolycaprolactone microspheres containing 1%, 2%, 5% or 10% paclitaxelinto phosphate buffered saline at 37° C. FIG. 16B is a photograph whichshows a CAM treated with control microspheres. FIG. 16C is a photographwhich shows a CAM treated with 5% paclitaxel loaded microspheres.

[0044]FIGS. 17A and 17B, respectively, are two graphs which show therelease of paclitaxel from EVA films, and the percent paclitaxelremaining in those same films over time. FIG. 17C is a graph which showsthe swelling of EVA/F127 films with no paclitaxel over time. FIG. 17D isa graph which shows the swelling of EVA/Span 80 films with no paclitaxelover time. FIG. 17E is a graph which depicts a stress vs. strain curvefor various EVA/F127 blends.

[0045]FIGS. 18A and 18B are two graphs which show the melting point ofPCL/MePEG polymer blends as a function of % MePEG in the formulation(18A), and the percent increase in time needed for PCL paste at 60° C.to being to solidify as a function of the amount of MePEG in theformulation (18B). FIG. 18C is a graph which depicts the softness ofvarying PCL/MePEG polymer blends. FIG. 18D is a graph which shows thepercent weight change over time for polymer blends of various MePEGconcentrations. FIG. 18E is a graph which depicts the rate of paclitaxelrelease over time from various polymer blends loaded with 1% paclitaxel.FIGS. 18F and 18G are graphs which depict the effect of varyingquantities of paclitaxel on the total amount of paclitaxel released froma 20% MePEG/PCL blend. FIG. 18H is a graph which depicts the effect ofMePEG on the tensile strength of a MePEG/PCL polymer.

[0046]FIG. 19A is a photograph which shows control (unloaded)thermopaste on a CAM. Note that both large vessels and small vessels(capillaries) are found immediately adjacent to the paste. Blood flow inthe area around and under the paste is unaffected. FIG. 19B is aphotograph of 20% paclitaxel-loaded thermopaste on a CAM. Note thedisruption of the vasculature when compared to the surrounding tissues.The drug loaded paste has blocked the growth of the capillaries, causedregression of the larger vessels, and created a region of avascularityon the CAM assay. FIG. 19C is a photograph of 0.5% paclitaxel-loadedthermopaste on a CAM (Mag.—40×). Briefly, the paclitaxel-loadedthermopaste disk induced an avascular zone measuring 6 mm in diameter onthe CAM. This avascular region was induced by blocking new capillarygrowth and occluding, disrupting, and regressing the existing bloodvessels found within the treated region. FIG. 19D is a photograph ofcontrol (unloaded) Thermopaste on a CAM. Briefly, after a 2 dayexposure, the blood vessel organization of the CALM (Mag=50×) treatedwith the control paste shows normal blood vessel organization.Functional vessels are located immediately adjacent to the unloadedpaste.

[0047]FIGS. 20A and 20B are two photographs of a CAM having a tumortreated with control (unloaded) thermopaste. Briefly, in FIG. 20 A thecentral white mass is the tumor tissue. Note the abundance of bloodvessels entering the tumor from the CAM in all directions. The tumorinduces the ingrowth of the host vasculature through the production of“angiogenic factors.” The tumor tissue expands distally along the bloodvessels which supply it. FIG. 20B is an underside view of the CAM shownin 20A. Briefly, this view demonstrates the radial appearance of theblood vessels which enter the tumor like the spokes of a wheel. Notethat the blood vessel density is greater in the vicinity of the tumorthan it is in the surrounding normal CAM tissue. FIGS. 20C and 20D aretwo photographs of a CAM having a tumor treated with 20%paclitaxel-loaded thermopaste. Briefly, in FIG. 20C the central whitemass is the tumor tissue. Note the paucity of blood vessels in thevicinity of the tumor tissue. The sustained release of the angiogenesisinhibitor is capable of overcoming the angiogenic stimulus produced bythe tumor. The tumor itself is poorly vascularized and is progressivelydecreasing in size. FIG. 20D is taken from the underside of the CAMshown in 20C, and demonstrates the disruption of blood flow into thetumor when compared to control tumor tissue. Note that the blood vesseldensity is reduced in the vicinity of the tumor and is sparser than thatof the normal surrounding CAM tissue.

[0048]FIG. 21A is a graph which shows the effect of paclitaxel/PCL ontumor growth. FIGS. 21B and 21C are two photographs which show theeffect of control, 10%. and 20% paclitaxel-loaded thermopaste on tumorgrowth.

[0049]FIG. 22A is a photograph of synovium from a PBS injected joint.FIG. 22B is a photograph of synovium from a microsphere injected joint.FIG. 22C is a photograph of cartilage from joints injected with PBS, andFIG. 22D is a photograph of cartilage from joints injected withmicrospheres.

[0050]FIG. 23A is a photograph of a 0.3% Paclitaxel Ophthalmic DropSuspension on a CAM (Mag.=32×). The plastic ring was used to localizethe drug treatment to the CAM. Note the lack of blood vessels locatedwithin and immediately adjacent to the ring. The functional bloodvessels bordering the avascular zone are defined by their “elbowing”morphology away form the drug source. FIG. 23B is a photograph of acontrol (unloaded) Ophthalmic Drop Suspension on a CAM (Mag=32×). Notethe normal organization of the CAM blood vessels and the abundance offunctional vessels located within the ring.

[0051]FIG. 24A is a photograph of a 2.5% Paclitaxel-Loaded Stent Coating(Mag=26×). Briefly, the blood vessels surrounding the avascular zone aremorphologically redirected away from the paclitaxel source: thisproduces an avascular zone which can measure up to 6 mm in diameter. Thedisrupted vascular remnants which represent vascular regression can beseen within the avascular zone. FIG. 24B is a control (unloaded) StentCoating (Mag=26×). Briefly, the blood vessels of the CAM are foundimmediately adjacent to the stent and do not illustrate anymorphological alterations.

[0052]FIG. 25 is a photograph of a control stent. Briefly, this imageshows the longitudinal orientation of a nylon stent incorporated withingliosarcoma tissue of the rat liver. Ingrowth within the nylon stent isevident.

[0053]FIG. 26 is a photograph of a control stent. Briefly, this imagealso illustrates tumor ingrowth within the lumen of the nylon stent.

[0054]FIG. 27 is a photograph of a lung. Briefly, in addition to largeliver tumors, metastasis to the lung is common. Such metastases areevident by the presence of small white lobules seen throughout the lung.

[0055]FIG. 28A is a photograph of Suramin and Cortisone Acetate on a CAM(Mag=8×). Briefly, this image shows an avascular zone treated with 20 μgof suramin and 70 μg of cortisone acetate in 0 5% methylcellulose. Notethe blood vessels located at the periphery of the avascular zone whichare being redirected away from the drug source. FIG. 28B is a photographwhich shows the vascular detail of the effected region at a highermagnification (Mag=20×). Note the avascular regions and the typical“elbowing” effect of the blood vessels bordering the avascular zone.

[0056]FIG. 29A is a graph which shows the chemiluminescence response ofneutrophils (5×10⁶ cells/ml) to plasma opsonized CPPD crystals (50mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 4.5 μM, (Δ.) 14μM, (▴) 28 μM, (□) 46 μM; n=3. FIG. 29B is a graph which shows the timecourse concentration dependence of paclitaxel inhibition of plasmaopsonized CPPD crustal induced neutrophil chemiluminescence.

[0057]FIG. 30A is a graph which shows superoxide anion production byneutrophils (5×10⁶ cells/mi) in response to plasma opsonized CPPDcrystals (50 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28μM, (Δ) Control (cells alone); n=3. FIG. 30B is a graphic which showsthe time course concentration dependence of paclitaxel inhibition ofplasma opsonized CPPD crustal induced neutrophil superoxide anionproduction; n 3.

[0058]FIG. 31A is a graph which shows the chemiluminescence response ofneutrophils (5×10⁶ cells/ml) in response to plasma opsonized zymozan (1mg/ml). Effect of paclitaxel at (o) no drug, () 28 μM; n=3. FIG. 31B isa graph which shows plasma opsonized zymosan induced neutrophilsuperoxide anion production. Effect of paclitaxel at (o) no paclitaxel,() 28 μM, (Δ) Control (cells alone); n 3.

[0059]FIG. 32A is a graph which shows myeloperoxidase release fromneutrophils (5×10⁶ cells/mi) in response to plasma opsonized CPPDcrystals (50 mg/ml). Effect of paclitaxel at (o) no paclitaxel, () 28μM. (Δ) Control (cells alone), (▴) Control (cells with paclitaxel at 28μM); n=3.

[0060]FIG. 32B is a graph which shows the concentration dependence ofpaclitaxel inhibition of myeloperoxidase release from neutrophils inresponse to plasma opsonized CPPD crystals; n 3.

[0061]FIG. 33 is a graph which shows lysozyme release from neutrophils(5×10⁶/ml) in response to plasma opsonized CPPD crystals (50 mg/ml).Effect of paclitaxel at (o) no paclitaxel, () 28 μM (Δ) Control (cellsalone). (▴) Control (cells and paclitaxel at 28 μM); n 3.

[0062]FIG. 34 is a graph which depicts proliferation of synoviocytes atvarious concentrations of paclitaxel.

[0063]FIG. 35 is a bar graph which depicts the cytotoxicity ofpaclitaxel at various concentrations to proliferating synoviocytes.

[0064]FIGS. 36A, 36B, and 36C are photographs of a series of gels whichshow the effect of various concentrations of paclitaxel on c-FOSexpression.

[0065]FIGS. 37A and 37B are photographs of a series oft gels which showthe effect of various concentrations of paclitaxel on collagenaseexpression.

[0066]FIG. 38 is a bar graph which depicts the effects of paclitaxel onviability of normal chondrocytes in vitro.

[0067]FIG. 39 is a graph which shows the percentage of paclitaxelrelease based upon gelatinized-paclitaxel of either a large (7200 μm) orsmall (2100 μm) size.

[0068]FIG. 40 is a graph which shows the effect of gelatin and/or sodiumchloride on the release of paclitaxel from PCL.

[0069]FIG. 41 is a graph which shows the release of paclitaxel fromPDLLA-PEG-PDLLA cylinders containing 20% paclitaxel.

[0070]FIG. 42A is a graph which depicts the time course of paclitaxelrelease from 2.5 mg pellets of PCL. FIG. 42B is a graph which shows thepercent paclitaxel remaining in the pellet, over time.

[0071]FIG. 43A is a graph which shows the effect of MePEG on paclitaxelrelease from PCL paste leaded with 20% paclitaxel. FIG. 43B is a graphwhich shows the percent paclitaxel remaining in the pellet, over time.

[0072]FIGS. 44A and 44B are graphs which show the effect of variousconcentrations of MePEG in PCL in terms of melting point (44A) and timeto solidify (44B).

[0073]FIG. 45 is a graph which shows the effect of MePEG incorporationinto PCL on the tensile strength and time to fail of the polymer.

[0074]FIG. 46 is a graph which shows the effect of irradiation onpaclitaxel release.

[0075]FIGS. 47A, B C, D and E show the effect of MTX release from PCLover time.

[0076]FIG. 48 is a graph of particle diameter (μm) determined by aCoulter® LS130 Particle Size Analysis.

[0077]FIG. 49 is a graph of particle diameter (μm) determined by aCoulter® LS130 Particle Size Analysis.

[0078]FIG. 50 is a graph which shows paclitaxel release from variouspolymeric formulations.

[0079]FIG. 51 is a graph which shows the effect of plasma opsonizationof polymeric microspheres on the chemiluminescence response ofneutrophils (20 mg/ml microspheres in 0.5 ml of cells (conc. 5×10⁶cells/ml) to PCL microspheres.

[0080]FIG. 52 is a graph which shows the effect of precoating plasma+/−2% pluronic F127 on the chemiluminescence response of neutrophils(5×10⁶ cells/ml) to PCL microspheres

[0081]FIG. 53 is a graph which shows the effect of precoating plasma+/−2% pluronic F127 on the chemiluminescence response of neutrophils(5×10⁶ cells/ml) to PMMA microspheres

[0082]FIG. 54 is a graph which shows the effect of precoating plasma+/−2% pluronic F127 on the chemiluminescence response of neutrophils(5×10⁶ cells/ml) to PLA microspheres

[0083]FIG. 55 is a graph which shows the effect of precoating plasma+/−2% pluronic F127 on the chemiluminescence response of neutrophils(5×10⁶ cells/ml) to EVA:PLA microspheres

[0084]FIG. 56 is a graph which shows the effect of precoating IgG (2mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescenceresponse of neutrophils to PCL microspheres.

[0085]FIG. 57 is a graph which shows the effect of precoating IgG (2mg/ml ), or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescenceresponse of neutrophils to PMMA microspheres.

[0086]FIG. 58 is a graph which shows the effect of precoating IgG (2mg/ml), or 2% pluronic F127 then IgG (2 ml/ml) on the chemiluminescenceresponse of neutrophils to PVA microspheres.

[0087]FIG. 59 is a graph which shows the effect of precoating IgG (2mg/ml), or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescenceresponse of neutrophils to EVA.PLA microspheres.

[0088]FIG. 60 is a photograph of 10% methotrexate (“MTX”) loadedmicrospheres made from a 50:50 ratio of PLA:GA (IV 0.78).

[0089]FIG. 61 is a graph which depicts the release of 10% loaded vanadylsulfate from PCL.

[0090]FIG. 62 is a photograph of hyaluronic acid microspheres containingvanadium sulfate.

[0091]FIG. 63A is a graph which depicts the release of organic vanadatefrom PCL. FIG. 63B depicts the percentage of organic vanadate remainingover a time course.

[0092]FIG. 64 is a photograph showing poly D.L. lactic acid microspherescontaining organic vanadate.

[0093]FIGS. 65A and 65B are photographs of control (uncoated) stentswhich show typical epithelial ingrowth seen at both 8 weeks (A) and at16 weeks (B). Indentations of the stent tines (t) and narrowing of thelumen (lu) are shown. There is progressive epithelial overgrowth of thestent surface over this time by fibrous and inflammatory tissue.

[0094]FIGS. 66A, 66B, 66C, and 66D are a series of photographs whichshow control and paclitaxel-coated biliary stents. FIG. 66A illustratesthe obliteration of the stent lumen by the process of benign epithelialovergrowth. At higher magnification (66B), the fibrous and inflammatorytissue is evident with little luminal space remaining. Thepaclitaxel-treated biliary duct remains patent (66C). At highermagnification, normal biliary tract epithelium is present with onlyminimal alteration of the mucosal lining by the coated stent tines (t).

DETAILED DESCRIPTION OF THE INVENTION

[0095] As noted above, the present invention provides methods andcompositions which utilize anti-angiogenic factors. Briefly, within thecontext of the present invention, anti-angiogenic factors should beunderstood to include any protein, peptide, chemical, or other molecule,which acts to inhibit vascular growth. A variety of methods may bereadily utilized to determine the anti-angiogenic activity of a givenfactor, including for example, chick chorioallantoic membrane (“CAM”)assays. Briefly, as described in more detail below in Examples 2A and2C, a portion of the shell from a freshly fertilized chicken egg isremoved, and a methyl cellulose disk containing a sample of theanti-angiogenic factor to be tested is placed on the membrane. Afterseveral days (e.g., 48 hours), inhibition of vascular growth by thesample to be tested may be readily determined by visualization of thechick chorioallantoic membrane in the region surrounding the methylcellulose disk. Inhibition of vascular growth may also be determinedquantitatively, for example, by determining the number and size of bloodvessels surrounding ,he methyl cellulose disk, as compared to a controlmethyl cellulose disk. Although anti-angiogenic factors as describedherein are considered to inhibit the formation of new blood vessels ifthey do so in merely a statistically significant manner, as compared toa control, within preferred aspects such anti-angiogenic factors willcompletely inhibit the formation of new blood vessels, as well as reducethe size and number of previously existing vessels.

[0096] In addition to the CAM assay described above, a variety of otherassays may also be utilized to determine the efficacy of anti-angiogenicfactors in vivo, including for example, mouse models which have beendeveloped for this purpose (see Roberston et al., Cancer. Res.51:1339-1344, 1991). In addition, a variety of representative in vivoassays relating to various aspects of the inventions described hereinhave also been described in more detail below in Examples 5 to 7, and 17to 19.

[0097] As noted above, the present invention provides compositionscomprising an anti-angiogenic factor, and a polymeric carrier. Briefly,a wide variety of anti-angiogenic factors may be readily utilized withinthe context of the present invention. Representative examples includeAnti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel,Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1, PlasminogenActivator Inhibitor-2, and various forms of the lighter “d group”transition metals. These and other anti-angiogenic factors will bediscussed in more detail below.

[0098] Briefly, Anti-Invasive Factor, or “AIF” which is prepared fromextracts of cartilage, contains constituents which are responsible forinhibiting the growth of new blood vessels. These constituents comprisea family of 7 low molecular weight proteins (<50,000 daltons) (Kuettnerand Pauli, “Inhibition of neovascularization by a cartilage factor” inDevelopment of the Vascular System, Pitman Books (CIBA FoundationSymposium 100), pp. 163-173, 1983), including a variety of proteinswhich have inhibitory effects against a variety of proteases (Eisenteinet al, Am. J. Pathol. 81:337-346, 1975; Langer et al., Science193:70-72, 1976: and Horton et al., Science 199: 1342-1345, 1978). AIFsuitable for use within the present invention may be readily preparedutilizing techniques known in the art (e.g., Eisentein et al, supra;Kuettner and Pauli, supra; and Langer et al.. supra). Purifiedconstituents of MEF such as Cartilage-Derived Inhibitor (“CDI”) (seeMoses et al., Science 248: 1048-1410, 1990) may also be readily preparedand utilized within the context of the present invention.

[0099] Retinoic acids alter the metabolism of extracellular matrixcomponents, resulting in the inhibition of angiogenesis. Addition ofproline analogs, angiostatic steroids, or heparin may be utilized inorder to synergistically increase the anti-angiogenic effect oftransretinoic acid. Retinoic acid, as well as derivatives thereof whichmay also be utilized in the context of the present invention, may bereadily obtained from commercial sources, including for example. SigmaChemical Co. (#R2625).

[0100] Paclitaxel is a highly derivatized diterpenoid (Wani et al., J.Am. Chem. Soc. 93:2325, 1971) which has been obtained from the harvestedand dried bark of Taxus brevifolia (Pacific Yew.) and TaxomycesAndreanae and Eudophytic Fungus of the Pacific Yew (Stierle et al.,Science 60:214-216, 1993). Generally, paclitaxel acts to stabilizemicrotubular structures by binding tubulin to form abnormal mitoticspindles. “Paclitaxel” (which should be understood herein to includeanalogues and derivatives such as, for example. TAXOL®, TAXOTERE®,10-desacetyl analogues of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogues of paclitaxel) may be readily prepared utilizingtechniques known to those skilled in the art (see also WO 94/07882, WO94/07881, WO 94/07880, WO 94,/07876. WO 93/23555, WO 93/10076, U.S. Pat.Nos. 5,294,637, 5,283,523, 5,279,949, 5,274,137, 5,202,448, 5,200,534,5,229,529, and EP 590267), or obtained from a variety of commercialsources, including for example, Sigma Chemical Co. St. Louis, Mo.(T7402—from Taxus brevifolia).

[0101] Suramin is a polysulfonated naphthylurea compound that istypically used as a trypanocidal agent. Briefly, Suramin blocks thespecific cell surface binding of various growth factors such as plateletderived growth factor (“PDGF”), epidermal growth factor (“EGF”),transforming growth factor (“TGF-β”), insulin-like growth factor(“IGF-1”), and fibroblast growth factor (“βFGF”). Suramin may beprepared in accordance with known techniques, or readily obtained from avariety of commercial sources, including for example Mobay Chemical Co.,New York. (see Gagliardi et al., Cancer Res. 52:5073-5075, 1992; andCoffey, Jr, et al., J. of Cell. Phys. 132:143-148, 1987).

[0102] Tissue Inhibitor of Metalloproteinases-1 (“TIMP”) is secreted byendothelial cells which also secrete MTPases. TIMP is glycosylated andhas a molecular weight of 28 5 kDa. TIMP-1 regulates angiogenesis bybinding to activated metalloproteinases, thereby suppressing theinvasion of blood vessels into the extracellular matrix. TissueInhibitor of Metalloproteinases-2 (“TIMP”-“2”) may also be utilized toinhibit angiogenesis. Briefly, TIMP-2 is a 21 kDa nonglycosylatedprotein which binds to metalloproteinases in both the active and latent,proenzyme forms. Both TIMP-1 and TIMP-2 may be obtained from commercialsources such as Synergen, Boulder, Colo.

[0103] Plasminogen Activator Inhibitor-1 (PA) is a 50 kDa glycoproteinwhich is present in blood platelets, and can also be synthesized byendothelial cells and muscle cells. PAI-1 inhibits t-PA and urokinaseplasminogen activator at the basolateral site of the endothelium, andadditionally regulates the fibrinolysis process. Plasminogen ActivatorInhibitor-2 (PAI-2) is generally found only in the blood under certaincircumstances such as in pregnancy, and in the presence or tumors.Briefly, PAI-2 is a 56 kDa protein which is secreted by monocytes andmacrophages. It is believed to regulate fibrinolytic activity, and inparticular inhibits urokinase plasminogen activator and tissueplasminogen activator, thereby preventing fibrinolysis.

[0104] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0105] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate (i.e., VO₃—) and orthovanadate(i.e.. VO₄ ³—) complexes such as, for example, ammonium metavanadate(i.e., NH₄VO₃). sodium metavanadate (i.e., NaVO₃), and sodiumorthovanadate (i.e., Na₃VO₄). Suitable vanadvl (i.e., VO²⁺) complexesinclude, for example, vanadyl acetylacetonate and vanadyl sulfateincluding vanadyl sulfate hydrates such as vanadyl sulfate mono- andtrihydrates.

[0106] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate (i.e., WO₄ ²⁻)complexes include ammonium tungstate (i.e., (NE₄)₂WO₄), calciumtungstate (i.e., CaWO₄), sodium tungstate dihydrate (i.e., Na₂WO₄.2H₂O),and tungstic acid (i.e., H₂WO₄). Suitable tungsten oxides includetungsten (IV) oxide (i.e., WO₂) and tungsten (VI) oxide (i.e., WO₃).Suitable oxo molybdenum complexes include molybdate, molybdenum oxide,and molybdenyl complexes. Suitable molybdate (i.e., MoO₄ ²⁻)) complexesinclude ammonium molybdate (i.e., (NH₄)₂MoO₄) and its hydrates. sodiummolybdate (i.e., Na₇MoO₄) and its hydrates, and potassium molybdate(i.e., K₂MoO₄) and its hydrates. Suitable molybdenum oxides includemolybdenum (VI) oxide (i.e., MoO₂), molybdenum (VI) oxide (i.e., MoO₃),and molybdic acid. Suitable molybdenyl (i.e., MoO₂ ²⁻) complexesinclude, for example, molybdenyl acetylacetonate. Other suitabletungsten and molybdenum complexes include hydroxo derivatives derivedfrom, for example, glycerol, tartaric acid, and sugars.

[0107] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include Platelet Factor 4 (Sigma Chemical Co.. #F1385);Protamine Sulphate (Clupeine) (Sigma Chemical Co., #P4505); SulphatedChitin Derivatives (prepared from queen crab shells), (Sigma ChemicalCo., #C3641, Murata et al, Cancer Res. 51:22-26, 1991); SulphatedPolysaccharide Peptidoglycan Complex (SP-PG) (the function of thiscompound may be enhanced by the presence of steroids such as estrogen,and tamoxifen citrate); Staurosporine (Sigma Chemical Co., #S4400);Modulators of Matrix Metabolism, including for example, proline analogs[[(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co.. #A0760)),cishydroxyproline. d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265),Thiaproline (Sigma Chemical Co., #T0631)], α,α-dipyridyl (Sigma ChemicalCo. #D7505), β-aminopropionitrile fumarate (Sigma Chemical Co.,#A3134)]}; MDL 27032 (4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; MerionMerrel Dow Research Institute); Methotrexate (Sigma Chemical Co.,#A6770; Hirata et al., Arthritis and Rheumatism 32:1065-1073, 1989);Mitoxantrone (Polverini and Novak, Biochem. Biophys. Res. Comm.140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985; SigmaChemical Co., #P8754); Interferons (e.g., Sigma Chemical Co., 13265) 2Macroglobulin-serum (Sigma Chemical Co., #M7151); ChIMP-3 (Pavloff etal., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Sigma ChemicalCo., #C7268; Tomkinson et al., Biochem J. 286:475-480, 1992);β-Cyclodextrin Tetradecasulfate (Sigma Chemical Co., .#C4767);Eponemycin; Camptothecin; Fumagillin (Sigma Chemical Co., #F6771;Canadian Patent No. 2,024,306; Ingber et al., Nature 348:555-557, 1990);Gold Sodium Thiomalate (“GST”; Sigma:G4022; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); (D-Penicillamine (“CDPT”; Sigma ChemicalCo., #P4875 or P5000(HCI)); β-1-anticollagenase-serum; α2-antiplasmin(Sigma Chem. Co.:A0914; Holmes et al., J. Biol. Chem. 262(4):1659-1664,1987); Bisantrene (National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole: metalloproteinaseinhibitors such as BB94 and the peptide CDPGYIGSR-NH₂ (SEQUENCE IDNO. 1) (Iwaki Glass, Tokyo, Japan).

[0108] Although the above anti-angiogenic factors have been provided forthe purposes of illustration, it should be understood that the presentinvention is not so limited. In particular, although certainanti-angiogenic factors are specifically referred to above, the presentinvention should be understood to include analogues, derivatives andconjugates of such anti-angiogenic factors For example, paclitaxelshould be understood to refer to not only the common chemicallyavailable form of paclitaxel, but analogues (e.g., taxotere, as notedabove) and paclitaxel conjugates (e.g., paclitaxel-PEG,paclitaxel-dextran, or paclitaxel-xylos).

[0109] Anti-angiogenic compositions of the present invention mayadditionally comprise a wide variety of compounds in addition to theanti-angiogenic factor and polymeric carrier. For example,anti-angiogenic compositions of the present invention may also, withincertain embodiments of the invention, also comprise one or moreantibiotics, anti-inflammatories, anti-viral agents, anti-fungal agentsand/or anti-protozoal agents. Representative examples of antibioticsincluded within the compositions described herein include: penicillins;cephalosporins such as cefadroxil, cefazolin, cefaclor; aminoglycosidessuch as gentamycin and tobramycin; sulfonamides such assulfamethoxazole; and metronidazole. Representative examples ofanti-inflammatories include: steroids such as prednisone, prednisolone,hydrocortisone, adrenocorticotropic hormone, and sulfasalazine; andnon-steroidal anti-inflammatory drugs (“NSAIDS”) such as aspirin,ibuprofen, naproxen, fenoprofen, indomethacin, and phenylbutazone.Representative examples of antiviral agents include acyclovir,ganciclovir, zidovudine. Representative examples of antifungal agentsinclude: nystatin, ketoconazole, griseofulvin, flucytosine, miconazole,clotrimazole. Representative examples of antiprotozoal agents include:pentamidine isethionate, quinine, chloroquine, and mefloquine.

[0110] Anti-angiogenic compositions of the present invention may alsocontain one or more hormones such as thyroid hormone, estrogen,progesterone, cortisone and/or growth hormone, other biologically activemolecules such as insulin, as well as T_(H)1 (e.g., Interleukins -2,-12, and -15, gamma interferon) or T_(H)2 (e.g., Interleukins - 4 and-10) cytokines.

[0111] Within certain preferred embodiments of the invention,anti-angiogenic compositions are provided which contain one or morecompounds which disrupt microtubule function. Representative examples ofsuch compounds include paclitaxel (discussed above). estramustine(available from Sigma; Wang and Stearns Cancer Res. 48:6262-6271, 1988),epothilone, curacin-A, colchicine, methotrexate, vinblastine and4-tert-butyl-[3-(2-chloroethyl)ureido]benzene (“tBCEU”)

[0112] Anti-angiogenic compositions of the present invention may alsocontain a wide variety of other compounds, including for example:α-adrenergic blocking agents, angiotensin II receptor antagonists andreceptor antagonists for histamine. serotonin, endothelin; inhibitors ofthe sodium/hydrogen antiporter (e.g., amiloride and its derivatives);agents that modulate intracellular Ca²⁺ transport such as L-type (e.g.diltiazem, nifedipine, verapamil) or T-type Ca²⁺ channel blockers (e.g.,amiloride), calmodulin antagonists (e.g., H₇) and inhibitors of thesodium/calcium antiporter (e.g., amiloride); ap-1 inhibitors (fortyrosine kinases, protein kinase C. myosin light chain kinase.Ca²⁺/calmodulin kinase II, casein kinase II); anti-depressants (e.g.amytriptyline, fluoxetine, LUVOX® and PAXIL®)); cytokine and/or growthfactors, as well as their respective receptors, (e.g., the interleukins,α, β or γ-IFN, GM-CSF, G-CSF, epidermal growth factor, transforminggrowth factors alpha and beta, TNF, and antagonists of vascularepithelial growth factor, endothelial growth factor, acidic or basicfibroblast growth factors, and platelet dervived growth factor);inhibitors of the IP₃ receptor (e.g., heparin); protease and collagenaseinhibitors (e.g. TIMPs, discussed above); nitrovasodilators (e.g.,isosorbide dinitrate); anti-mitotic agents (e.g., colchicine,anthracyclines and other antibiotics, folate antagonists and otheranti-metabolites, vinca alkaloids, nitrosoureas, DNA alkylating agents,topoisomerase inhibitors, purine antagonists and analogs, pyrimidineantagonists and analogs, alkyl sulfonates); immunosuppressive agents(e.g., adrenocorticosteroids, cyclosporine); sense or antisenseoligonucleotides (e.g., DNA, RNA, nucleic acid analogues (e.g., peptidenucleic acids) or any combinations of these); and inhibitors oftranscription factor activity (e.g., lighter d group transition metals).

[0113] Anti-angiogenic compositions of the present invention may alsocomprise additional ingredients such as surfactants (either hydrophilicor hydrophobic; see Example 13), anti-neoplastic or chemotherapeuticagents (e.g., 5-fluorouracil, vincristine, vinblastine, cisplatin,doxyrubicin, adriamycin, or tamocifen), radioactive agents (e.g., Cu-64,Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-109, In-111,I-123, I-125, I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211,Pb-212 and Bi-212) or toxins (e.g., ricin, abrin, diphtheria toxin,cholera toxin, crelonin, pokeweed antiviral protein, tritin, Shigellatoxin, and Pseudomonas exotoxin A).

[0114] As noted above, anti-angiogenic compositions of the presentinvention comprise an anti-angiogenic factor and a polymeric carrier. Inaddition to the wide array of anti-angiogenic factors and othercompounds discussed above, anti-angiogenic compositions of the presentinvention are provided in a wide variety of polymeric carriers,including for example both biodegradable and non-biodegradablecompositions. Representative examples of biodegradable compositionsinclude albumin, gelatin, starch, cellulose, dextrans, polysaccharides,fibrinogen, poly (D,L lactide), poly (D,L-lactide-co-glycolide), poly(glycolide), poly (hydroxybutyrate), poly (alkylcarbonate) and poly(orthoesters) (see generally Illum. L., Davids. S. S. (eds.) “Polymersin controlled Drug Delivery” Wright. Bristol. 1987; Arshady, J.Controlled Release 17 1-22, 1991. Pitt. Int. J. Phar. 59:173-196, 1990:Holland et al., J. Controlled Release 4:155-0180, 1986) Representativeexamples of nondegradable polymers include EVA copolymers, siliconerubber and poly (methylmethacrylate). Particularly preferred polymericcarriers include poly (ethylene-vinyl acetate)(40% cross-linked), poly(D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomersand polymers, poly (glycolic acid), copolymers of lactic acid andglycolic acid, poly (caprolactone), poly (valerolactone),polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid)with polyethylene glycol and blends thereof.

[0115] Polymeric carriers may be fashioned in a variety of forms,including for example, rod-shaped devices, pellets, slabs, or capsules(see, e.g., Goodell et al., Am. J. Hosp. Pharm. 43:1454-1461, 1986.Langer et al., “Controlled release of macromolecules from polymers”, inBiomedical polymers, Polymeric materials and pharmaceuticals forbiomedical use, Goldberg. E. P., Nakagim. A. (eds.) Academic Press, pp.113-137, 1980; Rhine et al. J. Pharm. Sci. 69:-65-270, 1980; Brown etal., J. Pharm. Sci. 72:1181-1185, 1983; and Bawa et al., J. ControlledRelease 1:259-267, 1985). Anti-angiogenic factors may be linked byocclusion in the matrices of the polymer, bound by covalent linkages, orencapsulated in microcapsules. Within certain preferred embodiments ofthe invention, anti-angiogenic compositions are provided in non-capsularformulations such as microspheres (ranging from nanometers tomicrometers in size), pastes, threads of various size, films and sprays.

[0116] Preferably, anti-angiogenic compositions of the present invention(which comprise one or more anti-angiogenic factors, and a polymericcarrier) are fashioned in a manner appropriate to the intended use.Within certain aspects of the present invention, the anti-angiogeniccomposition should be biocompatible, and release one or moreanti-angiogenic factors over a period of several days to months. Forexample, “quick release” or “burst” anti-angiogenic compositions areprovided that release greater than 10%, 20%, or 25%, (w/v) of ananti-angiogenic factor (e.g., paclitaxel) over a period of 7 to 10 days.Such “quick release” compositions should, within certain embodiments, becapable of releasing chemotherapeutic levels (where applicable) of adesired anti-angiogenic factor. Within other embodiments, “low release”anti-angiogenic compositions are provided that release less than 1%(w/v) of an anti-angiogenic factor over a period of 7 to 10 days.Further, anti-angiogenic compositions of the present invention shouldpreferably be stable for several months and capable of being producedand maintained under sterile conditions.

[0117] Within certain aspects of the present invention, anti-angiogeniccompositions may be fashioned in any size ranging from 50 nm to 500 μm,depending upon the particular use. For example, when used for thepurpose of tumor embolization (as discussed below), it is generallypreferable to fashion the anti-angiogenic composition in microspheres ofbetween 15 and 500 μm, preferably between 15 and 200 μm, and mostpreferably, between 25 and 150 μm. Alternatively, such compositions mayalso be readily applied as a “spray”, which solidifies into a film orcoating. Such sprays may be prepared from microspheres of a wide arrayof sizes, including for example, from 0.1 μm to 3 μm, from 10 μm to 30μm, and from 30 μm to 100 μm (see Example 8).

[0118] Anti-angiogenic compositions may also be prepared, given thedisclosure provided herein, for a variety of other applications. Forexample, for administration to the cornea, the anti-angiogenic factorsof the present invention may be incorporated into muco-adhesive polymers(e.g., polyacralic acids such as (CARBOPOL®, dextron, polymethacrylate,or starch (see LeYung and Robinson, J. of Controlled Rel. 5:223, 1988)),or nanometer-sized microspheres (see generally, Kreuter J. ControlledRelease 16:169-176, 1991. Couvreur and Vauthier, J. Controlled Release17:187-198, 1991).

[0119] Anti-angiogenic compositions of the present invention may also beprepared in a variety of “paste” or gel forms. For example, within oneembodiment of the invention, anti-angiogenic compositions are providedwhich are liquid at one temperature (e.g., temperature greater than 37°C., such as 40° C., 45>C., 50° C., 55° C. or 60° C.), and solid orsemi-solid at another temperature (e.g., ambient body temperature, orany temperature lower than 37° C.). Such “thermopastes” may be readilymade given the disclosure provided herein (see, e.g. Examples 10 and14).

[0120] Within yet other aspects of the invention, the anti-angiogeniccompositions of the present invention may be formed as a film.Preferably, such films are generally less than 5, 4, 3, 2, or 1, mmthick, more preferably less than 0.75 mm or 0.5 mm thick, and mostpreferably less than 500 μm to 100 μm thick. Such films are preferablyflexible with a good tensile strength (e.g., greater than 50, preferablygreater than 100, and more preferably greater than 150 or 200 N/cm²),good adhesive properties (i.e., readily adheres to moist or wetsurfaces). and has controlled permeability. Representative examples ofsuch films are set forth below in the Examples (see e.g., Example 13).

[0121] Representative examples of the incorporation of anti-angiogenicfactors such as those described above into a polymeric carriers isdescribed in more detail below in Examples 3, 4 and 8-15.

POLYMERIC CARRIERS FOR THE RELEASE OF HYDROPHOBIC COMPOUNDS

[0122] Within further aspects of the present invention, polymericcarriers are provided which are adapted to contain and release ahydrophobic compound, the carrier containing the hydrophobic compound incombination faith a carbohydrate, protein or polypeptide. Within certainembodiments, the polymeric carrier contains or comprises regions,pockets, or granules of one or more hydrophobic compounds. For example,within one embodiment of the invention, hydrophobic compounds may beincorporated within a matrix which contains the hydrophobic compound,followed by incorporation of the matrix within the polymeric carrier. Avariety of matrices can be utilized in this regard, including forexample, carbohydrates and polysaccharides such as starch, cellulose,dextran, methylcellulose, and hyaluronic acid, proteins or polypeptidessuch as albumin, collagen and gelatin (see e.g, Example 31). Withinalternative embodiments, hydrophobic compounds may be contained within ahydrophobic core, and this core contained within a hydrophilic shell.For example, as described in Example 38, paclitaxel may be incorporatedinto a hydrophobic core (e.g., of the poly D,L lactic acid-PEG or MePEGaggregate) which has a hydrophilic shell.

[0123] A wide variety of hydrophobic compounds may be released from thepolymeric carriers described above, including for example: certainhydrophobic compounds which disrupt microtubule function such aspaclitaxel and estramustine; hydrophobic proteins such as myelin basicprotein, proteolipid proteins of CNS myelin, hydrophobic cell wallprotein, porins, membrane proteins (EMBO J. 12(9):3409-3415, 1993),myelin oligodendrocyte glycoprotein (“MOG”) (Biochem. and Mol. Biol.Ant. 30(5):945-958, 1993, P27 Cancer Res. 53(17e ):4096-4101, 1913,bacterioopsin, human surfactant protein (“HSB”; J. Biol. Chem.268(15):11160-11166, 1993), and SP-B or SP-C (Biochimica et BiophysicaActa 1105(1):161-169, 1992).

ARTERIAL EMBOLIZATION

[0124] In addition to the compositions described above, the presentinvention also provides a variety of methods which utilize theabove-described anti-angiogenic compositions. In particular, within oneaspect of the present invention methods are provided for embolizing ablood vessel, comprising the step of delivering into the vessel atherapeutically effective amount of an anti-angiogenic composition (asdescribed above), such that the blood vessel is effectively occluded.Therapeutically effective amounts suitable for occluding blood vesselsmay be readily determined given the disclosure provided below, and asdescribed in Example 6. Within a particularly preferred embodiment, theanti-angiogenic composition is delivered to a blood vessel whichnourishes a tumor (see FIG. 13).

[0125] Briefly, there are a number of clinical situations (e.g.,bleeding, tumor development) where it is desirable to reduce or abolishthe blood supply to an organ or region. As described in greater detailbelow, this may be accomplished by injecting anti-angiogeniccompositions of the present invention into a desired blood vesselthrough a selectively positioned catheter (see FIG. 13). The compositiontravels via the blood stream until it becomes wedged in the vasculature,thereby physically (or chemically) occluding the blood vessel. Thereduced or abolished blood flow to the selected area results ininfarction (cell death due to an inadequate supply of oxygen andnutrients) or reduced blood loss from a damaged vessel.

[0126] For use in embolization therapy, anti-angiogenic compositions ofthe present invention are preferably non-toxic, thrombogenic, easy toinject down vascular catheters, radio-opaque, rapid and permanent ineffect, sterile, and readily available in different shapes or sizes atthe time of the procedure. In addition, the compositions preferablyresult in the slow (ideally, over a period of several weeks to months)release of an anti-angiogenic factor. Particularly preferredanti-angiogenic compositions should have a predictable size of 15-200 μmafter being injected into the vascular system. Preferably, they shouldnot clump into larger particles either in solution or once injected. Inaddition, preferable compositions should not change shape or physicalproperties during storage prior to use.

[0127] Embolization therapy may be utilized in at least three principalways to assist in the management of neoplasms: (1) definitive treatmentof tumors (usually benign); (2) for preoperative embolization; and (3)for palliative embolization. Briefly, benign tumors may sometimes besuccessfully treated by embolization therapy alone. Examples of suchtumors include simple tumors of vascular origin (e.g., haemangiomas),endocrine tumors such as parathyroid adenomas, and benign bone tumors.

[0128] For other tumors. (e.g. renal adenocarcinoma), preoperativeembolization may be employed hours or days before surgical resection inorder to reduce operative blood loss, shorten the duration of theoperation, and reduce the risk of dissemination of viable malignantcells by surgical manipulation of the tumor. Many tumors may besuccessfully embolized preoperatively, including for examplenasopharyngeal tumors, glomus jugular tumors, meningiomas,chemodectomas, and vagal neuromas.

[0129] Embolization may also be utilized as a primary mode of treatmentfor inoperable malignancies, in order to extend the survival time ofpatients with advanced disease. Embolization may produce a markedimprovement in the quality of life of patients with malignant tumors byalleviating unpleasant symptoms such as bleeding, venous obstruction andtracheal compression. The greatest benefit from palliative tumorembolization, however, may be seen in patients suffering from thehumoral effects of malignant endocrine tumors, wherein metastases fromcarcinoid tumors and other endocrine neoplasms such as insulinomas andglucagonomas may be slow growing, and yet cause great distress by virtueof the endocrine syndromes which they produce.

[0130] In general, embolization therapy utilizing anti-angiogeniccompositions of the present invention is typically performed in asimilar manner, regardless of the site. Briefly, angiography (a road mapof the blood vessels) of the area to be embolized is First performed byinjecting radiopaque contrast through a catheter inserted into an arteryor vein (depending on the site to be embolized) as an X-ray is taken.The catheter may be inserted either percutaneously or by surgery. Theblood vessel is then embolized by refluxins anti-angiogenic compositionsof the present invention through the catheter, until flow is observed tocease. Occlusion may be confirmed by repeating the angiogram.

[0131] Embolization therapy generally results in the distribution ofcompositions containing anti-angiogenic factors throughout theinterstices of the tumor or vascular mass to be treated. The physicalbulk of the embolic particles clogging the arterial lumen results in theocclusion of the blood supply. In addition to this effect, the presenceof an anti-angiogenic factor(s) prevents the formation of new bloodvessels to supply the tumor or vascular mass, enhancing the devitalizingeffect of cutting off the blood supply.

[0132] Therefore, it should be evident that a wide variety of tumors maybe embolized utilizing the compositions of the present invention.Briefly, tumors are typically divided into two classes: benign andmalignant. In a benign tumor the cells retain their differentiatedfeatures and do not divide in a completely uncontrolled manner. Inaddition, the tumor is localized and nonmetastatic. In a malignanttumor, the cells become undifferentiated, do not respond to the body'sgrowth and hormonal signals, and multiply in an uncontrolled manner; thetumor is invasive and capable of spreading to distant sites(metastasizing).

[0133] Within one aspect of the present invention, metastases (secondarytumors) of the liver may be treated utilizing embolization therapy.Briefly, a catheter is inserted via the femoral or brachial artery andadvanced into the hepatic artery by steering it through the arterialsystem under fluoroscopic guidance. The catheter is advanced into thehepatic arterial tree as far as necessary to allow complete blockage ofthe blood vessels supplying the tumor(s), while sparing as many of thearterial branches supplying normal structures as possible. Ideally thiswill be a segmental branch of the hepatic artery, but it could be thatthe entire hepatic artery distal to the origin of the gastroduodenalartery, or even multiple separate arteries, will need to be blockeddepending on the extent of tumor and its individual blood supply. Oncethe desired catheter position is achieved, the artery is embolized byinjecting anti-angiogenic compositions (as described above) through thearterial catheter until flow in the artery to be blocked ceases,preferably even after observation for 5 minutes. Occlusion of the arterymay be confirmed by injecting radiopaque contrast through the catheterand demonstrating by fluoroscopy or X-ray film that the vessel whichpreviously filled with contrast no longer does so. The same proceduremay be repeated with each feeding artery to be occluded.

[0134] As noted above, both benign and malignant tumors may be embolizedutilizing compositions of the present invention. Representative examplesof benign hepatic tumors include Hepatocellular Adenoma, CavernousHaemangioma, and Focal Nodular Hyperplasia. Other benign tumors, whichare more rare and often do not have clinical manifestations, may also betreated. These include Bile Duct Adenomas, Bile Duct Cystadenomas,Fibromas, Lipomas, Leiomyomas, Mesotheliomas, Teratomas, Myxomas, andNodular Regenerative Hyperplasia.

[0135] Malignant Hepatic Tumors are generally subdivided into twocategories: primary and secondary. Primary tumors arise directly fromthe tissue in which they are found. Thus, a primary liver tumor isderived originally from the cells which make up the liver tissue (suchas hepatocytes and biliary cells). Representative examples of primaryhepatic malignancies which may be treated by arterial embolizationinclude Hepatocellularcarcinoma, Cholangiocarcinoma, Angiosarcoma,Cystadenocarcinoma, Squamous Cell Carcinoma, and Hepatoblastoma.

[0136] A secondary tumor, or metastasis, is a tumor which originatedelsewhere in the body but has now spread to a distant organ. The commonroutes for metastasis are direct growth into adjacent structures, spreadthrough the vascular or lymphatic systems, and tracking along tissueplanes and body spaces (peritoneal fluid. cerebrospinal fluid, etc).Secondary hepatic tumors are one of the most common causes of death inthe cancer patient, and are by far and away the most common form ofliver tumor. Although virtually any malignancy can metastasize to theliver, tumors which are most likely to spread to the liver include:cancer of the stomach, colon, and pancreas; melanoma; tumors of thelung, oropharynx, and bladder, Hodgkin's and non-Hodgkin's lymphoma;tumors of the breast, ovary, and prostate. Each one of the above-namedprimary tumors has numerous different tumor types which may be treatedby arterial embolization (for example, there are over 32 different typesof ovarian cancer).

[0137] As noted above, embolization therapy utilizing anti-angiogeniccompositions of the present invention may also be applied to a varietyof other clinical situations where it is desired to occlude bloodvessels. Within one aspect of the present invention, arteriovenousmalformation may be treated by administration of one of theabove-described compositions. Briefly, arteriovenous malformations(vascular malformations) refers to a group of diseases wherein at leastone (and most typically, many) abnormal communications between arteriesand veins occur, resulting in a local tumor-like mass composedpredominantly of blood vessels. Such disease may be either congenital oracquired.

[0138] Within one embodiment of the invention, an arteriovenousmalformation may be treated by inserting a catheter via the femoral orbrachial artery, and advancing it into the feeding artery underfluoroscopic guidance. The catheter is preferably advanced as far asnecessary to allow complete blockage of the blood vessels supplying thevascular malformation, while sparing as many of the arterial branchessupplying normal structures as possible (ideally this will be a singleartery, but most often multiple separate arteries may need to beoccluded, depending on the extent of the vascular malformation and itsindividual blood supply). Once the desired catheter position isachieved, each artery may be embolized utilizing anti-angiogeniccompositions of the present invention.

[0139] Within another aspect of the invention, embolization may beaccomplished in order to treat conditions of excessive bleeding. Forexample, menorrhagia (excessive bleeding with menstruation) may bereadily treated by embolization of uterine arteries. Briefly, theuterine arteries are branches of the internal iliac arteriesbilaterally. Within one embodiment of the invention, a catheter may beinserted via the femoral or brachial artery, and advanced into eachuterine artery by steering it through the arterial system underfluoroscopic guidance. The catheter should be advanced as far asnecessary to allow complete blockage of the blood vessels to the uterus,while sparing as many arterial branches that arise from the uterineartery and supply normal structures as possible Ideally a single uterineartery on each side may be embolized, but occasionally multiple separatearteries may need to be blocked depending on the individual bloodsupply. Once the desired catheter position is achieved, each artery maybe embolized by administration of the anti-angiogenic compositions asdescribed above.

[0140] In a like manner, arterial embolization may be accomplished in avariety of other conditions, including for example, for acute bleeding,vascular abnormalities, central nervous system disorders, andhypersplenism.

USE OF ANTI-ANGIOGENIC COMPOSITIONS AS COATINGS FOR STENTS

[0141] As noted above, the present invention also provides stents,comprising a generally tubular structure (which includes for example,spiral shapes), the surface of which is coated with a composition asdescribed above. Briefly, a stent is a scaffolding, usually cylindricalin shape, that may be inserted into a body passageway (e.g., bile ducts)or a portion of a body passageway, which has been narrowed, irregularlycontured, obstructed, or occluded by a disease process (e.g., ingrowthby a tumor) in order to prevent closure or reclosure of the passaseway.Stents act by physically holding open the walls of the body passage intowhich they are inserted.

[0142] A variety of stents may be utilized within the context of thepresent invention, including for example, esophageal stents, vascularstents, biliary stents, pancreatic stents, ureteric and urethral stents,lacrimal stents, Eustachian tube stents, fallopian tube stents andtracheal/bronchial stents.

[0143] Stents may be readily obtained from commercial sources, orconstructed in accordance with well-known techniques. Representativeexamples of stents include those described in U.S. Pat. No. 4,768,523,entitled “Hydrogel Adhesive;” U.S. Pat. No. 4,776,337, entitled“Expandable Intraluminal Graft, and Method and Apparatus for Implantingand Expandable Intraluminal Graft;” U.S. Pat. No. 5,041,126 entitled“Endovascular Stent and Delivery System;” U.S. Pat. No. 5,052,998entitled “Indwelling Stent and Method of Use.” U.S. Pat. No. 5,064,435entitled “Self-Expanding Prosthesis Having Stable Axial Length;” U.S.Pat. No. 5,089,606, entitled “Water-insoluble Polysaccharide HydrogelFoam for Medical Applications;” U.S. Pat. No. 5,147,370, entitled“Nitinol Stent for Hollow Body Conduits,” U.S. Pat. No. 5,176,626,entitled “Indwelling Stent;” U.S. Pat. No. 5,213,580, entitled“Biodegradable polymeric Endoluminal Sealing Process;” and U.S. Pat. No.5,328,471, entitled “Method and Apparatus for Treatment of Focal Diseasein Hollow Tubular Organs and Other Tissue Lumens.”

[0144] Stents may be coated with anti-angiogenic compositions oranti-angiogenic factors of the present invention in a variety ofmanners, including for example: (a) by directly affixing to the stent ananti-angiogenic composition (e.g., by either spraying the stent with apolymer/drug film, or by dipping the stent into a polymer/drugsolution), (b) by coating the stent with a substance such as a hydrogelwhich will in turn absorb the anti-angiogenic composition (oranti-angiogenic factor above), (c) by interweaving anti-angiogeniccomposition coated thread (or the polymer itself formed into a thread)into the stent structure, (d) by inserting the stent into a sleeve ormesh which is comprised of or coated with an anti-angiogeniccomposition, or (e) constructing the stent itself with ananti-angiogenic composition. Within preferred embodiments of theinvention, the composition should firmly adhere to the stent duringstorage and at the time of insertion, and should not be dislodged fromthe stent when the diameter is expanded from its collapsed size to itsfull expansion size. The anti-angiogenic composition should alsopreferably not degrade during storage, prior to insertion, or whenwarmed to body temperature after expansion inside the body. In addition,it should preferably coat the stent smoothly and evenly, with a uniformdistribution of angiogenesis inhibitor, while not changing the stentcontour. Within preferred embodiments of the invention, theanti-angiogenic composition should provide a uniform, predictable,prolonged release of the anti-angiogenic factor into the tissuesurrounding the stent once it has been deployed. For vascular stents, inaddition to the above properties, the composition should not render thestent thrombogenic (causing blood clots to form), or cause significantturbulence in blood flow (more than the stent itself would be expectedto cause if it was uncoated).

[0145] Within another aspect of the present invention, methods areprovided for expanding the lumen of a body passageway, comprisinginserting a stent into the passageway, the stent having a generallytubular structure, the surface of the structure being coated with ananti-angiogenic composition (or, an anti-angiogenic factor alone), suchthat the passageway is expanded. A variety of embodiments are describedbelow wherein the lumen of a body passageway is expanded in order toeliminate a biliary, esophageal, tracheal/bronchial, urethral orvascular obstruction. In addition, a representative example is describedin more detail below in Example 7.

[0146] Generally, stents are inserted in a similar fashion regardless ofthe site or the disease being treated. Briefly, a preinsertionexamination, usually a diagnostic imaging procedure, endoscopy, ordirect visualization at the time of surgery, is generally firstperformed in order to determine the appropriate positioning for stentinsertion. A guidewire is then advanced through the lesion or proposedsite of insertion, and over this is passed a delivery catheter whichallows a stent in its collapsed form to be inserted. Typically, stentsare capable of being compressed, so that they can be inserted throughtiny cavities via small catheters, and then expanded to a largerdiameter once they are at the desired location. Once expanded, the stentphysically forces the walls of the passageway apart and holds them open.As such, they are capable of insertion via a small opening, and yet arestill able to hold open a large diameter cavity or passageway. The stentmay be self-expanding (e.g., the Wallstent and Gianturco stents),balloon expandable (e.g. the Palmaz stent and Strecker stent), orimplanted by a change in temperature (e.,., the Nitinol stent).

[0147] Stents are typically maneuvered into place under radiologic ordirect visual control, taking particular care to place the stentprecisely across the narrowing in the organ being treated. The deliverycatheter is then removed, leaving the stent standing on its own as ascaffold. A post insertion examination, usually an x-ray, is oftenutilized to confirm appropriate positioning.

[0148] Within a preferred embodiment of the invention, methods areprovided for eliminating biliary obstructions, comprising inserting abiliary stent into a biliary passageway, the stent having a generallytubular structure, the surface of the structure being coated with acomposition as described above, such that the biliary obstruction iseliminated. Briefly, tumor overgrowth of the common bile duct results inprogressive cholestatic jaundice which is incompatible with life.Generally, the biliary system which drains bile from the liver into theduodenum is most often obstructed by (1) a tumor composed of bile ductcells (cholangiocarcinoma), (2) a tumor which invades the bile duct(e.g., pancreatic carcinoma), or (3) a tumor which exerts extrinsicpressure and compresses the bile duct (e.g., enlarged lymph nodes).

[0149] Both primary biliary tumors, as well as other tumors which causecompression of the biliary tree may be treated utilizing the stentsdescribed herein. One example of primary biliary tumors areadenocarcinomas (which are also called Klatskin tumors when found at thebifurcation of the common hepatic duct). These tumors are also referredto as biliary carcinomas, choledocholangiocarcinomas, or adenocarcinomasof the biliary system. Benign tumors which affect the bile duct (e.g.,adenoma of the biliary system), and, in rare cases, squamous cellcarcinomas of the bile duct and adenocarcinomas of the gallbladder, mayalso cause compression of the biliary tree and therefore, result inbiliary obstruction.

[0150] Compression of the biliary tree is most commonly due to tumors ofthe liver and pancreas which compress and therefore obstruct the ducts.Most of the tumors from the pancreas arise from cells of the pancreaticducts. This is a highly fatal form of cancer (50% of all cancer deaths;26,000 new cases per year in the U.S.) with an average of 6 monthssurvival and a 1 year survival rate of only 10/o. When these tumors arelocated in the head of the pancreas they frequently cause biliaryobstruction, and this detracts significantly from the quality of life ofthe patient. While all types of pancreatic tumors are generally referredto as “carcinoma of the pancreas” there are histologic subtypesincluding: adenocarcinoma, adenosquamous carcinoma, cystadeno-carcinoma,and acinar cell carcinoma. Hepatic tumors, as discussed above, may alsocause compression of the biliary tree, and therefore cause obstructionof the biliary ducts.

[0151] Within one embodiment of the invention, a biliary stent is firstinserted into a biliary passageway in one of several ways: from the topend by inserting a needle through the abdominal wall and through theliver (a percutaneous transhepatic cholangiogram or “PTC”), from thebottom end by cannulating the bile duct through an endoscope insertedthrough the mouth, stomach, and duodenum (an endoscopic retrogradecholanqiogram or “ERCP”); or by direct incision during a surgicalprocedure. A preinsertion examination. PTC, ERCP, or directvisualization at the time of surgery should generally be performed todetermine the appropriate position for stent insertion. A guidewire isthen advanced through the lesion, and over this a delivery catheter ispassed to allow the stent to be inserted in its collapsed form. If thediagnostic exam was a PTC, the guidewire and delivery catheter isinserted via the abdominal wall, while if the original exam was an ERCPthe stent may be placed via the mouth. The stent is then positionedunder radiologic, endoscopic, or direct visual control taking particularcare to place it precisely across the narrowing in the bile duct. Thedelivery catheter is then removed leaving the stent standing as ascaffolding which holds the bile duct open. A further cholangiogram maybe performed to document that the stent is appropriately positioned.

[0152] Within yet another embodiment of the invention, methods areprovided for eliminating esophageal obstructions, comprising insertingan esophageal stent into an esophagus, the stent having a generallytubular structure, the surface of the structure being coated with ananti-angiogenic composition as described above, such that the esophagealobstruction is eliminated. Briefly, the esophagus is the hollow tubewhich transports food and liquids from the mouth to the stomach. Cancerof the esophagus or invasion by cancer arising in adjacent organs (e.g.cancer of the stomach or lung) results in the inability to swallow foodor saliva. Within this embodiment, a preinsertion examination, usually abarium swallow or endoscopy should generally be performed in order todetermine the appropriate position for stent insertion. A catheter orendoscope may then be positioned through the mouth, and a guidewire isadvanced through the blockade. A stent delivery catheter is passed overthe guidewire under radiologic or endoscopic control, and a stent isplaced precisely across the narrowing in the esophagus. A post insertionexamination, usually a barium swallow x-ray, may be utilized to confirmappropriate positioning.

[0153] Within other embodiments of the invention, methods are providedfor eliminating tracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi, the stent having agenerally tubular structure, the surface of which is coated with ananti-angiogenic composition as described above, such that thetracheal/bronchial obstruction is eliminated. Briefly, the trachea andbronchi are tubes which carry air from the mouth and nose to the lungs.Blockage of the trachea by cancer, invasion by cancer arising inadjacent organs (e.g., cancer of the lung), or collapse of the tracheaor bronchi due to chondromalacia (weakening of the cartilage rings)results in inability to breathe. Within this embodiment of theinvention, preinsertion examination, usually an endoscopy, shouldgenerally be performed in order to determine the appropriate positionfor stent insertion. A catheter or endoscope is then positioned throughthe mouth, and a guidewire advanced through the blockage. A deliverycatheter is then passed over the guidewire in order to allow a collapsedstent to be inserted. The stent is placed under radiologic or endoscopiccontrol in order to place it precisely across the narrowing. Thedelivery catheter may then be removed leaving the stent standing as ascaffold on its own. A post insertion examination, usually abronchoscopy may be utilized to confirm appropriate positioning.

[0154] Within another embodiment of the invention, methods are providedfor eliminating urethral obstructions, comprising inserting a urethralstent into a urethra, the stent having a generally tubular structure,the surface of the structure being coated with an anti-angiogeniccomposition as described above, such that the urethral obstruction iseliminated. Briefly, the urethra is the tube which drains the bladderthrough the penis. Extrinsic narrowing of the urethra as it passesthrough the prostate, due to hypertrophy of the prostate, occurs invirtually every man over the age of 60 and causes progressive difficultywith urination. Within this embodiment, a preinsertion examination,usually an endoscopy or urethrogram should generally first be performedin order to determine the appropriate position for stent insertion,which is above the external urinary sphincter at the lower end, andclose to flush with the bladder neck at the upper end. An endoscope orcatheter is then positioned through the penile opening and a guidewireadvanced into the bladder. A delivery catheter is then passed over theguidewire in order to allow stent insertion. The delivery catheter isthen removed, and the stent expanded into place. A post insertionexamination, usually endoscopy or retrograde urethrogram, may beutilized to confirm appropriate position.

[0155] Within another embodiment of the invention, methods are providedfor eliminating vascular obstructions, comprising inserting a vascularstent into a blood vessel, the stent having a generally tubularstructure, the surface of the structure being coated with ananti-angiogenic composition as described above, such that the vascularobstruction is eliminated. Briefly, stents may be placed in a wide arrayof blood vessels, both arteries and veins, to prevent recurrent stenosisat the site of failed angioplasties, to treat narrowings that wouldlikely fail if treated with angioplasty, and to treat post surgicalnarrowings (e.g. dialysis graft stenosis). Representative examples ofsuitable sites include the iliac, renal, and coronary arteries, thesuperior vena cava, and in dialysis grafts. Within one embodiment,angiography is first performed in order to localize the site forplacement of the stent. This is typically accomplished by injectingradiopaque contrast through a catheter inserted into an artery or veinas an x-ray is taken. A catheter may then be inserted eitherpercutaneously or by surgery into the femoral artery, brachial artery,femoral vein, or brachial vein, and advanced into the appropriate bloodvessel by steering it through the vascular system under fluoroscopicguidance. A stent may then be positioned across the vascular stenosis. Apost insertion angiogram may also be utilized in order to confirmappropriate positioning.

USE OF ANTI-ANGIOGENIC COMPOSITIONS IN SURGICAL PROCEDURES

[0156] As noted above, anti-angiogenic compositions may be utilized in awide variety of surgical procedures. For example, within one aspect ofthe present invention an anti-angiogenic compositions (in the form of,for example, a spray or film) may be utilized to coat or spray an areaprior to removal of a tumor, in order to isolate normal surroundingtissues from malignant tissue, and/or to prevent the spread of diseaseto surrounding tissues Within other aspects of the present invention,anti-angiogenic compositions (e.g., in the form of a spray) may bedelivered via endoscopic procedures in order to coat tumors, or inhibitangiogenesis in a desired locale. Within yet other aspects of thepresent invention, surgical meshes which have been coated withanti-angiogenic compositions of the present invention may be utilized inany procedure wherein a surgical mesh might be utilized. For example,within one embodiment of the invention a surgical mesh ladened with ananti-angiogenic composition may be utilized during abdominal cancerresection surgery (e.g., subsequent to colon resection) in order toprovide support to the structure, and to release an amount of theanti-angiogenic factor.

[0157] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administering ananti-angiogenic composition as described above to the resection marginsof a tumor subsequent to excision, such that the local recurrence ofcancer and the formation of new blood vessels at the site is inhibited.Within one embodiment of the invention, the anti-angiogeniccomposition(s) (or anti-angiogenic factor(s) alone) are administereddirectly to the tumor excision site (e.g., applied by swabbing, brushingor otherwise coating the resection margins of the tumor with theanti-angiogenic composition(s) or factor(s)). Alternatively, theanti-angiogenic composition(s) or factor(s) may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compositionsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

[0158] Within one aspect of the present invention, anti-angiogeniccompositions (as described above) may be administered to the resectionmargin of a wide variety of tumors, including for example, breast,colon, brain and hepatic tumors. For example, within one embodiment ofthe invention, anti-angiogenic compositions may be administered to thesite of a neurological tumor subsequent to excision, such that theformation of new blood vessels at the site are inhibited. Briefly, thebrain is highly functionally localized; i.e., each specific anatomicalregion is specialized to carry out a specific function. Therefore it isthe location of brain pathology that is often more important than thetype. A relatively small lesion in a key area can be far moredevastating than a much larger lesion in a less important area.Similarly, a lesion on the surface of the brain may be easy to resectsurgically, while the same tumor located deep in the brain may not (onewould have to cut through too many vital structures to reach it). Also,even benign tumors can be dangerous for several reasons: they may growin a key area and cause significant damage; even though they would becured by surgical resection this may not be possible; and finally, ifleft unchecked they can cause increased intracranial pressure. The skullis an enclosed space incapable of expansion. Therefore, if something isgrowing in one location, something else must be being compressed inanother location—the result is increased pressure in the skull orincreased intracranial pressure If such a condition is left untreated,vital structures can be compressed, resulting in death. The incidence ofCNS (central nervous system) malignancies is 8-16 per 100,000. Theprognosis of primary malignancy of the brain is dismal, with a mediansurvival of less than one year, even following surgical resection. Thesetumors, especially gliomas, are predominantly a local disease whichrecur within 2 centimeters of the original focus of disease aftersurgical removal.

[0159] Representative examples of brain tumors which may be treatedutilizing the compositions and methods described herein include GlialTumors (such as Anaplastic Astrocytoma, Glioblastoma Multiform,Pilocytic Astrocytoma, Oligodendroglioma, Ependymoma, MyxopapillaryEpendymoma, Subependymoma, Choroid Plexus Papilloma); Neuron Tumors(e.g., Neuroblastoma, Ganglioneuroblastoma, Ganglioneuroma, andMedulloblastoma); Pineal Gland Tumors (e.g., Pineoblastoma andPineocytoma); Menigeal Tumors (e.g., Meningioma, MeningealHemangiopericytoma, Meningeal Sarcoma); Tumors of Nerve Sheath Cells(e.g., Schwannoma (Neurolemmoma) and Neurofibroma); Lymphomas (e.g.,Hodgkin's and Non-Hodgkin's Lymphoma (including numerous subtypes, bothprimary and secondary); Malformative Tumors (e.g., Craniopharyngioma,Epidermoid Cysts, Dermoid Cysts and Colloid Cysts); and MetastaticTumors (which can be derived from virtually any tumor, the most commonbeing from lung, breast, melanoma, kidney, and gastrointestinal tracttumors).

INFLAMMATORY ARTHRITIS

[0160] Inflammatory arthritis is a serious health problems in developedcountries, particularly given the increasing number of aged individuals.For example, one form of inflammatory arthritis, rheumatoid arthritis(RA) is a multisystem chronic, relapsing, inflammatory disease ofunknown cause. Although many organs can be affected, RA is basically asevere form of chronic synovitis that sometimes leads to destruction andankylosis of affected joints (taken from Robbinns Pathological Basis ofDisease, by R. S. Cotran. V Kumar, and S L. Robbins, W. B. Saunders Co.,1989). Pathologically the disease is characterized by a markedthickening of the synovial membrane which forms villous projections thatextend into the joint space, multilayering of the synoviocyte lining(synoviocyte proliferation), infiltration of the synovial membrane withwhite blood cells (macrophages, lymphocytes, plasma cells, and lymphoidfollicles; called an “inflammatory synovitis”), and deposition of fibrinwith cellular necrosis within the synovium. The tissue formed as aresult of this process is called pannus and eventually the pannus growsto fill the joint space. The pannus develops an extensive network of newblood vessels through the process of angiogenesis which is essential tothe evolution of the synovitis. Release of digestive enzymes [matrixmetalloproteinases (e.g., collagenase, stromelysin)] and other mediatorsof the inflammatory process (e.g., hydrogen peroxide, superoxides,lysosomal enzymes, and products of arachadonic acid metabolism) From thecells of the pannus tissue leads to the progressive destruction of thecartilage tissue. The pannus invades the articular cartilage leading toerosions and fragmentation of the cartilage tissue. Eventually there iserosion of the subchondral bone with fibrous ankylosis and ultimatelybony ankylosis, of the involved joint.

[0161] It is generally believed, but not conclusively proven, that RA isan autoimmune disease, and that many different arthriogenic stimuliactivate the immune response in the immunogenetically susceptible host.Both exogenous infectious agents (Ebstein-Barr Virus, Rubella virus,Cytomegalovirus, Herpes Virus, Human T-cell Lymphotropic Virus,Mycoplasma, and others) and endogenous proteins (collagen,proteoglycans, altered immunoglobulins) have been implicated as thecausative agent which triggers an inappropriate host immune response.Regardless of the inciting agent, autoimmunity plays a role in theprogression of the disease. In particular, the relevant antigen isingested by antigen-presenting cells (macrophages or dendritic cells inthe synovial membrane), processed, and presented to T lymphocytes. The Tcells initiate a cellular immune response and stimulate theproliferation and differentiation of B lymphocytes into plasma cells.The end result is the production of an excessive inappropriate immuneresponse directed against the host tissues [e.g., antibodies directedagainst Type II collagen, antibodies directed against the Fc portion ofautologous IgG (called “Rheumatoid Factor”)]. This further amplifies theimmune response and hastens the destruction of the cartilage tissue.Once this cascade is initiated numerous mediators of cartilagedestruction are responsible for the progression of rheumatoid arthritis.

[0162] Thus, within one aspect of the present invention, methods areprovided for treating or preventing inflammatory arthritis (e.g.,rheumatoid arthritis) comprising the step of administering to a patienta therapeutically effective amount of an anti-angiogenic factor oranti-angiogenic composition to a joint. Within a preferred embodiment ofthe invention, anti-angiogenic factors (including anti-angiogeniccompositions, as described above) may be administered directly byintra-articular injection, as a surgical paste, or as an oral agent(e.g., containing the anti-angiogenic factor thalidomide). Onerepresentative example of such a method is set forth in more detailbelow in Example 19.

[0163] As utilized within the context of the present invention, itshould be understood that efficatious administration of theanti-angiogenic factors and compositions described herein may beassessed in several ways. including: (1) by preventing or lessening thepathological and/or clinical symptoms associated with rheumatoidarthritis; (2) by downregulating the white blood cell response whichinitiates the inflammatory cascade and results in synovitis, swelling,pain, and tissue destruction; (3) by inhibiting the “tumor-like”proliferation of synoviocytes that leads to the development of a locallyinvasive and destructive pannus tissue; (4) by decreasing theproduction/activity of matrix metalloproteinases produced by white bloodcells, synoviocytes, chondrocytes, and endothelial cells, which degradethe cartilage matrix and result in irreversible destruction of thearticular cartilage; and (5) by inhibiting blood vessel formation whichprovides the framework and nutrients necessary for the growth anddevelopment of the pannus tissue. Furthermore, the anti-angiogenicfactors or compositions should not be toxic to normal chondrocytes attherapeutic levels. Each of these aspects will be discussed in moredetail below.

[0164] A. Inflammatory Response

[0165] Neutrophils are found in abundance in the synovial fluid, butonly in small numbers in the synovial membrane itself It is estimatedthat more than 1 billion neutrophils enter a moderately inflamedrheumatoid knee joint each day (Hollingsworth et al., 1967) and remainthere because no pathway exists by which they can leave the joint. Thesecells release reactive free radicals and lysosomal enzymes which degradethe cartilage tissue. Other PMN products such as prostaglandins andleukotrienes augment the inflammatory response and recruit moreinflammatory cells into the joint tissue.

[0166] Lymphocytes, particularly T cells, are present in abundance inthe diseased synovial tissue. Activated T cells produce a variety oflymphokines and cooperate with B cells to produce autoantibodies. Tcells products result in the activation macrophages, a cell which isthought to have an important role in the pathology of the disease. Themacrophages produce a variety destructive lysosomal enzymes,prostaglandins, and monokines and are also capable of stimulatingangiogenesis. One of the more important monokines secreted bymacrophages is IL-1 Briefly, IL-1 is known to: stimulate synthesis andrelease of collagenase by synoviocytes and synovial fibroblasts, inhibitproteoglycan synthesis by chondrocytes, activate osteoclasts, inducechanges in the endothelium of the synovial vasculature [stimulation ofendothelial production of plasminogen activator and colony stimulatingfactor, expression of leukocyte adhesion molecules, promotion ofprocoagulant activity (Wider et al., 1991)], and act as achemoattractant for lymphocytes and neutrophils.

[0167] Within one embodiment, downregulation of the white blood cellresponse, or inhibition of the inflammatory response, may be assessed bydetermination of the effect of the anti-angiogenic factor oranti-angiogenic composition on the response of neutrophils stimulatedwith opsonized CPPD crystals or opsonized zyrosan. Such methods areillustrated in more detail below in Example 22.

[0168] B. Synoviocyte Hyperplasia

[0169] During the development of R the synovial lining cells becomeactivated by products of inflammation or through phagocytosis of immunecomplexes. Several subtypes of synovial lining cells have beenidentified and all of them become intensely activated and undergoexcessive hyperplasia and growth when stimulated. As the synovial tissueorganizes to form a pannus, the number of synoviocytes, blood vessels,connective tissue elements, and inflammatory cells increases to form amass 100 times its original size. In many ways, the synovitis inrheumatoid arthritis behaves much like a localized neoplasia (Harris.1990). In fact, cultured rheumatoid synovial cells develop thephenotypic characteristics of anchorage-independent growth usuallyassociated with neoplastic cells if they given sufficientplateletderived growth factor (Lafyatis et al., 1989). In addition, thesynoviocytes also produce large amounts of collagenase, stromelysin,prostaglandins, and Interleukin-1.

[0170] The tumor-like proliferation of the cells of the synovialconnective tissue stroma (synoviocytes, fibroblast-like cells andneovascular tissue) produces a pannus with many features of a localizedmalignancy. Supporting this tumor analogy are several findings: thepannus expresses high levels of oncoproteins such as c-myc and c-fos,produces metalloproteinases to facilitate surrounding tissue invasion,express cytoskeletal markers characteristic of poorly differentiatedmesenchymal tissue (e.g., vimentin); synoviocytes in vitro grow rapidly,do not contact inhibit, form foci, and can be grown underanchorage-independent conditions in soft agarose, and pannus tissue iscapable of inducing the growth of a supporting vasculature (i.e.angiogenesis). All these findings are suggestive of a tissue in whichnormal growth regulation as been lost.

[0171] Within one embodiment, inhibition of synoviocyte proliferationmay be determined by, for example, analysis of ³H-thymidineincorporation into synoviocytes, or in vitro synoviocyte proliferation.Such methods are illustrated in more detail below in Example 23.

[0172] C. Matrix Metalloproteinases (MMP)

[0173] Irreparable degradation of the cartilage extracellular matrix isbelieved to be largely due to the enzymatic action of matrixmetalloproteinases on the components of the cartilage matrix. Althoughnumerous other enzymes are likely involved in the development of RA,collagenase (MMP-1) and stromelysin (MMP-3) play an important role(Vincetti et al., 1994) in disease progression. These enzymes arecapable of degrading type 11 collagen and proteoglycans respectively;the 2 major extracellular components of cartilage tissue. Cytokines suchas IL-1 epidermal growth factor (EGF), platelet-derived growth factor,and tumor necrosis factor are all potent stimulators of collagenase andstromelysin production. As described above, numerous cell types found inthe arthritic joint (white blood cells, synoviocytes, endothelial cells,and chondrocytes) are capable of synthesizing and secreting MMPS.

[0174] In proliferating rheumatoid synovial tissue, collagenase andstromelysin become the major gene products of the pannus and maycomprise as much as 2% of the messenger RNAs produced by the synovialfibroblasts (Brinkerhoff and Auble, 1990). Increased levels ofcollagenase and stromelysin are present in the cartilage of patientswith RA and the level of enzyme activity in the joint correlates wellwith the severity of the lesion (Martel-Pelletier et al., 1993;Walakovitis et al., 1992). Because these enzymes are fundamental to thepathology of RA and result in irreversible cartilage damage, manytherapeutic strategies have been devised to inhibit their effects.

[0175] Numerous naturally present inhibitors of MMP activity have beenidentified and named “TIMPS” for Tissue Inhibitors ofMetalloproteinases. Many of these protein inhibitors bind with theactive MMPs to form 1:1 noncovalent complexes which inactivate the MMPenzymes. The MMPs are produced locally by chondrocytes and synovialfibroblasts and are likely responsible for the normal regulation ofconnective tissue degradation. It is thought that much of the damage tothe cartilage matrix is due to a local imbalance between MMP and TIMPactivity. This is probably due to increased production ofmetalloproteinases while the production of TIMPs remains at a normal orconstant level (Vincetti et al., 1994). To overcome this, therapeuticstrategies have been designed to add exogenous TIMPs (e.g., thechemically modified tetracycline molecules, collagen substrateanalogues) or to upregulate TIMP production (retinoids, transforminggrowth factor β, IL-6, IL-11, oncostatin M) in an effort to restore theenzymatic balance. However this approach has yet to translate intosignificant clinical results.

[0176] An alternative approach is to inhibit or downregulate theproduction of the MMPs to restore a normal balance of activity.Naturally occurring compounds (TNFβ, all-trans retinoic acid) andsynthetic compounds (retinoids, glucocorticoid hormones) have beendemonstrated to inhibit MMP activity by suppressing transcription andsynthesis of these proteins. A post-transcriptional method of blockingMMP release could also be expected to result in a decrease in the amountof MMP produced and an improved balance between MMP and TIMP activity inthe joint.

[0177] Within one embodiment, a decrease in the production or activityof MMP's may be determined by, for example, analysis of IL-1 inducedcollagenase expression. One such method is illustrated in more detailbelow in Example 24.

[0178] D. Angiogenesis

[0179] The development of an extensive network of new blood vessels isessential to the development of the synovitis present in rheumatoidarthritis (Harris, 1990; Folkman et al., 1989; Sano et al., 1990).Several local mediators such as plateletderived growth factor (PDGF),TGF-β, and fibroblast growth factor (FGF) are likely responsible for theinduction and perpetuation of neovascularization within the synovium.Pannus tissue composed of new capillaries and synovial connective tissueinvades and destroys the articular cartilage. The migrating angiogenicvessels themselves produce and secrete increased levels ofmetalloproteinases such as collagenase and stromelysin capable ofdegrading the cartilage matrix (Case et al., 1989). The newly formedvessels are also quite “leaky” with gaps present between themicrovascular endothelial cells. This facilitates the exudation ofplasma proteins into the synovium (which increases swelling), enhancesWBCs movement from the circulation into the pannus tissue (whichincreases inflammation), and leads to the perivascular accumulation ofmononuclear inflammatory cells (Wilder et al., 1991).

[0180] In summary, the endothelial tissue plays an important role in thedevelopment of this disease by expressing the necessary surfacereceptors to allow inflammatory cells to leave the circulation and enterthe developing pannus, secreting proteolytic enzymes capable ofdegrading the cartilage matrix, and proliferating to form the newvessels (angiogenesis) required for the pannus tissue to increase insize and invade adjacent tissues.

[0181] Within one embodiment, inhibition of new blood vessel formationmay be readily determined in a variety of assays, including the CASTassay described above and within Example 2.

NEOVASCULAR DISEASES OF THE EYE

[0182] As noted above, the present invention also provides methods fortreating neovascular diseases of the eye, including for example, cornealneovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroblasia and macular degeneration.

[0183] Briefly, corneal neovascularization as a result of injury to theanterior segment is a significant cause of decreased visual acuity andblindness, and a major risk factor for rejection of corneal allografts.As described by Burger et al., Lab. Invest. 48:169-180, 1983, cornealangiogenesis involves three phases: a pre-vascular latent period, activeneovascularization, and vascular maturation and regression. The identityand mechanism of various angiogenic factors, including elements of theinflammatory response, such as leukocytes, platelets, cytokines, andeicosanoids, or unidentified plasma constituents have yet to berevealed.

[0184] Currently no clinically satisfactory therapy exists forinhibition of corneal neovascularization or regression of existingcorneal new vessels. Topical corticosteroids appear to have someclinical utility, presumably by limiting stromal inflammation.

[0185] Thus, within one aspect of the present invention methods areprovided for treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of an anti-angiogenic composition (as described above)to the cornea, such that the formation of blood vessels is inhibited.Briefly, the cornea is a tissue which normally lacks blood vessels. Incertain pathological conditions however, capillaries may extend into thecornea from the pericorneal vascular plexus of the limbus. When thecornea becomes vascularized, it also becomes clouded, resulting in adecline in the patient's visual acuity. Visual loss may become completeif the cornea completely opacitates.

[0186] Blood vessels can enter the cornea in a variety of patterns anddepths, depending upon the process which incites the neovascularization.These patterns have been traditionally defined by ophthalmologists inthe following types, pannus trachomatosus, pannus leprosus, pannusphylctenulosus, pannus degenerativus, and glaucomatous pannus. Thecorneal stroma may also be invaded by branches of the anterior ciliaryartery (called interstitial vascularization) which causes severaldistinct clinical lesions: terminal loops, a “brush-like” pattern, anumbel form, a lattice form, interstitial arcades (from episcleralvessels), and aberrant irregular vessels.

[0187] A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g. graft rejection and Stevens-Johnson'ssyndrome), alkali burns, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

[0188] While the cause of corneal neovascularization may vary, theresponse of the cornea to the insult and the subsequent vascularingrowth is similar regardless of the cause. Briefly, the location ofthe injury appears to be of importance as only those lesions situatedwithin a critical distance of the limbus will incite an angiogenicresponse. This is likely due to the fact that the angiogenic factorsresponsible for eliciting the vascular invasion are created at the siteof the lesion, and must diffuse to the site of the nearest blood vessels(the limbus) in order to exert their effect. Past a certain distancefrom the limbus, this would no longer be possible and the limbicendothelium would not be induced to grow into the cornea. Severalangiogenic factors are likely involved in this process, many of whichare products of the inflammatory response. Indeed, neovascularization ofthe cornea appears to only occur in association with an inflammatorycell infiltrate, and the degree of angiogenesis is proportional to theextent of the inflammatory reaction. Corneal edema further facilitatesblood vessel ingrowth by loosening the corneal stromal framework andproviding a pathway of “least resistance” through which the capillariescan grow.

[0189] Following the initial inflammatory reaction, capillary growthinto the cornea proceeds in the same manner as it occurs in othertissues. The normally quiescent endothelial cells of the limbiccapillaries and venules are stimulated to divide and migrate. Theendothelial cells project away from their vessels of origin, digest thesurrounding basement membrane and the tissue through which they willtravel, and migrate towards the source of the angiogenic stimulus. Theblind ended sprouts acquire a lumen and then anastomose together to formcapillary loops. The end result is the establishment of a vascularplexus within the corneal stroma.

[0190] Anti-angiogenic factors and compositions of the present inventionare useful by blocking the stimulatory effects of angiogenesispromoters, reducing endothelial cell division, decreasing endothelialcell migration, and impairing the activity of the proteolytic enzymessecreted by the endothelium.

[0191] Within particularly preferred embodiments of the invention, ananti-angiogenic factor may be prepared for topical administration insaline (combined with any of the preservatives and antimicrobial agentscommonly used in ocular preparations), and administered in eyedrop form.The anti-angiogenic factor solution or suspension may be prepared in itspure form and administered several times daily. Alternatively,anti-angiogenic compositions, prepared as described above, may also beadministered directly to the cornea. Within preferred embodiments, theanti-angiogenic composition is prepared with a muco-adhesive polymerwhich binds to cornea. Within further embodiments, the anti-angiogenicfactors or anti-angiogenic compositions may be utilized as an adjunct toconventional steroid therapy.

[0192] Topical therapy may also be useful prophylactically in corneallesions which are known to have a high probability of inducing anangiogenic response (such as chemical burns). In these instances thetreatment, likely in combination with steroids, may be institutedimmediately to help prevent subsequent complications.

[0193] Within other embodiments, the anti-angiogenic compositionsdescribed above may be injected directly into the corneal stroma by anophthalmologist under microscopic guidance. The preferred site ofinjection may vary with the morphology of the individual lesion, but thegoal of the administration would be to place the composition at theadvancing front of the vasculature (i.e., interspersed between the bloodvessels and the normal cornea). In most cases this would involveperilimbic corneal injection to “protect” the cornea from the advancingblood vessels. This method may also be utilized shortly after a cornealinsult in order to prophylactically prevent corneal neovascularization.In this situation the material could be injected in the perilimbiccornea interspersed between the corneal lesion and its undesiredpotential limbic blood supply. Such methods may also be utilized in asimilar fashion to prevent capillary invasion of transplanted corneas.In a sustained-release form injections might only be required 2-3 timesper year. A steroid could also be added to the injection solution toreduce inflammation resulting from the injection itself

[0194] Within another aspect of the present invention, methods areprovided for treating neovascular glaucoma, comprising the step ofadministering to a patient a therapeutically effective amount of ananti-angiogenic composition to the eye, such that the formation of bloodvessels is inhibited.

[0195] Briefly, neovascular glaucoma is a pathological condition whereinnew capillaries develop in the iris of the eye. The angiogenesis usuallyoriginates from vessels located at the pupillary margin, and progressesacross the root of the iris and into the trabecular meshwork.Fibroblasts and other connective tissue elements are associated with thecapillary growth and a fibrovascular membrane develops which spreadsacross the anterior surface of the iris. Eventually this tissue reachesthe anterior chamber angle where it forms synechiae. These synechiae inturn coalesce, scar, and contract to ultimately close off the anteriorchamber angle. The scar formation prevents adequate drainage of aqueoushumor through the angle and into the trabecular meshwork, resulting inan increase in intraocular pressure that may result in blindness.

[0196] Neovascular glaucoma generally occurs as a complication ofdiseases in which retinal ischemia is predominant. In particular, aboutone third of the patients with this disorder have diabetic retinopathyand 28% have central retinal vein occlusion. Other causes includechronic retinal detachment, end-stage glaucoma, carotid arteryobstructive disease, retrolental fibroplasia, sickle-cell anemia.intraocular tumors, and carotid cavernous fistulas. In its early stages,neovascular glaucoma may be diagnosed by high magnification slitlampbiomicroscopy, where it reveals small, dilated, disorganized capillaries(which leak fluorescein) on the surface of the iris. Later gonioscopydemonstrates progressive obliteration of the anterior chamber angle byfibrovascular bands. While the anterior chamber angle is still open,conservative therapies may be of assistance. However, once the anglecloses surgical intervention is required in order to alleviate thepressure.

[0197] Therefore, within one embodiment of the invention anti-angiogenicfactors (either alone or in an anti-angiogenic composition, as describedabove) may be administered topically to the eye in order to treat earlyforms of neovascular glaucoma.

[0198] Within other embodiments of the invention, anti-angiogeniccompositions may be implanted by injection of the composition into theregion of the anterior chamber angle. This provides a sustainedlocalized increase of anti-angiogenic factor, and prevents blood vesselgrowth into the area. Implanted or injected anti-angiogenic compositionswhich are placed between the advancing capillaries of the iris and theanterior chamber angle can “defend” the open angle fromneovascularization. As capillaries will not grow within a significantradius of the anti-angiogenic composition, patency of the angle could bemaintained. Within other embodiments, the anti-angiogenic compositionmay also be placed in any location such that the anti-angiogenic factoris continuously released into the aqueous humor. This would increase theanti-angiogenic factor concentration within the humor, which in turnbathes the surface of the iris and its abnormal capillaries, therebyproviding another mechanism by which to deliver the medication. Thesetherapeutic modalities may also be useful prophylactically and incombination with existing treatments.

[0199] Within another aspect of the present invention, methods areprovided for treating proliferative diabetic retinopathy, comprising thestep of administering to a patient a therapeutically effective amount ofan anti-angiogenic composition to the eyes, such that the formation ofblood vessels is inhibited.

[0200] Briefly, the pathology of diabetic retinopathy is thought to besimilar to that described above for neovascular glaucoma. In particular,background diabetic retinopathy is believed to convert to proliferativediabetic retinopathy under the influence of retinal hypoxia. Generally,neovascular tissue sprouts from the optic nerve (usually within 10 mm ofthe edge), and from the surface of the retina in regions where tissueperfusion is poor. Initially the capillaries grow between the innerlimiting membrane of the retina and the posterior surface of thevitreous. Eventually, the vessels grow into the vitreous and through theinner limiting membrane. As the vitreous contracts, traction is appliedto the vessels, often resulting in shearing of the vessels and blindingof the vitreous due to hemorrhage. Fibrous traction from scarring in theretina may also produce retinal detachment.

[0201] The conventional therapy of choice is panretinal photocoagulationto decrease retinal tissue, and thereby decrease retinal oxygen demands.Although initially effective, there is a high relapse rate with newlesions forming in other parts of the retina. Complications of thistherapy include a decrease in peripheral vision of up to 50% ofpatients, mechanical abrasions of the cornea, laser-induced cataractformation, acute glaucoma, and stimulation of subretinal neovasculargrowth (which can result in loss of vision). As a result, this procedureis performed only when several risk factors are present, and therisk-benefit ratio is clearly in favor of intervention.

[0202] Therefore, within particularly preferred embodiments of theinvention, proliferative diabetic retinopathy may be treated byinjection of an anti-angiogenic factor(s) (or anti-angiogeniccomposition) into the aqueous humor or the vitreous, in order toincrease the local concentration of anti-angiogenic factor in theretina. Preferably, this treatment should be initiated prior to theacquisition of severe disease requiring photocoagulation. Within otherembodiments of the invention, arteries which feed the neovascularlesions may be embolized (utilizing anti-angiogenic compositions, asdescribed above)

[0203] Within another aspect of the present invention, methods areprovided for treating retrolental fibroblasia, comprising the step ofadministering to a patient a therapeutically effective amount of ananti-angiogenic factor (or anti-angiogenic composition) to the eye, suchthat the formation of blood vessels is inhibited.

[0204] Briefly, retrolental fibroblasia is a condition occurring inpremature infants who receive oxygen therapy The peripheral retinalvasculature, particularly on the temporal side, does not become fullyformed until the end of fetal life. Excessive oxygen (even levels whichwould be physiologic at term) and the formation of oxygen free radicalsare thought to be important by causing damage to the blood vessels ofthe immature retina. These vessels constrict, and then becomestructurally obliterated on exposure to oxygen. As a result, theperipheral retina fails to vascularize and retinal ischemia ensues. Inresponse to the ischemia, neovascularization is induced at the junctionof the normal and the ischemic retina.

[0205] In 75% of the cases these vessels regress spontaneously. However,in the remaining 250% there is continued capillary growth, contractionof the fibrovascular component, and traction on both the vessels and theretina. This results in vitreous hemorrhage and/or retinal detachmentwhich can lead to blindness. Neovascular angle-closure glaucoma is alsoa complication of this condition

[0206] As it is often impossible to determine which cases willspontaneously resolve and which will progress in severity, conventionaltreatment (i.e., surgery) is generally initiated only in patients withestablished disease and a well developed pathology. This “wait and see”approach precludes early intervention, and allows the progression ofdisease in the 25% who follow a complicated course. Therefore, withinone embodiment of the invention, topical administration ofanti-angiogenic factors (or anti-angiogenic compositions, as describedabove) may be accomplished in infants which are at high risk fordeveloping this condition in an attempt to cut down on the incidence ofprogression of retrolental fibroplasia. Within other embodiments,intravitreous injections and/or intraocular implants of ananti-angiogenic composition may be utilized. Such methods areparticularly preferred in cases of established disease, in order toreduce the need for surgery.

OTHER THERAPEUTIC USES OF ANTI-ANGIOGENIC COMPOSITIONS

[0207] Anti-angiogenic factors and compositions of the present inventionmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. For example, anti-angiogenicfactors or compositions described herein may be formulated for topicaldelivery, in order to treat cancers such as skin cancer, head and necktumors, breast tumors, and Kaposi's sarcoma. Within yet other aspects,the anti-angiogenic factors or compositions provided herein may beutilized to treat superficial forms of bladder cancer by, for example,intravesical administration.

[0208] In addition to cancer, however, numerous other non-tumorigenicangiogenesis-dependent diseases which are characterized by the abnormalgrowth of blood vessels may also be treated with the anti-angiogenicfactors or compositions of the present invention. Representativeexamples of such non-tumorigenic angiogenesis-dependent diseases includehypertrophic scars and keloids, proliferative diabetic retinopathy(discussed above), rheumatoid arthritis (discussed above), arteriovenousmalformations (discussed above), atherosclerotic plaques, delayed woundhealing, hemophilic joints, nonunion fractures, Osler-Weber syndrome,psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia(discussed above) and vascular adhesions.

[0209] For example, within one aspect of the present invention methodsare provided for treating hypertrophic scars and keloids, comprising thestep of administering one of the above-described anti-angiogeniccompositions to a hypertrophic scar or keloid.

[0210] Briefly, healing of wounds and scar formation occurs in threephases: inflammation, proliferation, and maturation. The first phase,inflammation, occurs in response to an injury which is severe enough tobreak the skin. During this phase, which lasts 3 to 4 days, blood andtissue fluid form an adhesive coagulum and fibrinous network whichserves to bind the wound surfaces together. This is then followed by aproliferative phase in which there is ingrowth of capillaries andconnective tissue from the wound edges, and closure of the skin defect.Finally, once capillary and fibroblastic proliferation has ceased, thematuration process begins wherein the scar contracts and becomes lesscellular, less vascular, and appears flat and white. This final phasemay take between 6 and 12 months.

[0211] If too much connective tissue is produced and the wound remainspersistently cellular, the scar may become red and raised. If the scarremains within the boundaries of the original wound it is referred to asa hypertrophic scar, but if it extends beyond the original scar and intothe surrounding tissue, the lesion is referred to as a keloid.Hypertrophic scars and keloids are produced during the second and thirdphases of scar formation. Several wounds are particularly prone toexcessive endothelial and fibroblastic proliferation, including burns,open wounds, and infected wounds. With hypertrophic scars, some degreeof maturation occurs and gradual improvement occurs. In the case ofkeloids however, an actual tumor is produced which can become quitelarge. Spontaneous improvement in such cases rarely occurs.

[0212] Therefore, within one embodiment of the present invention eitheranti-angiogenic factors alone, or anti-angiogenic compositions asdescribed above, are directly injected into a hypertrophic scar orkeloid, in order to prevent the progression of these lesions. Thefrequency of injections will depend upon the release kinetics of thepolymer used (if present), and the clinical response. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development.

[0213] As noted above, within yet another aspect of the presentinvention, vascular grafts are provided comprising a synthetic tube, thesurface of which is coated with an anti-angiogenic composition asdescribed above. Briefly, vascular grafts are synthetic tubes, usuallymade of Dacron or Gortex, inserted surgically to bypass arterialblockages, most frequently from the aorta to the temoral, or the femoralto the popliteal artery. A major problem which particularly complicatesfemoral-popliteal bypass grafts is the formation of a subendothelialscar-like reaction in the blood vessel wall called neointimalhyperplasia, which narrows the lumen within and adjacent to either endof the graft, and which can be progressive. A graft coated with orcontaining anti-angiogenic factors (or anti-angiogenic compositions, asdescribed above) may be utilized to limit the formation of neointimalhyperplasia at either end of the graft. The graft may then be surgicallyplaced by conventional bypass techniques.

[0214] Anti-angiogenic compositions of the present invention may also beutilized in a variety of other manners. For example, they may beincorporated into surgical sutures in order to prevent stitchgranulomas, implanted in the uterus (in the same manner as an IUD) forthe treatment of menorrhagia or as a form of female birth control,administered as either a peritoneal lavage fluid or for peritonealimplantation in the treatment of endometriosis, attached to a monoclonalantibody directed against activated endothelial cells as a form ofsystemic chemotherapy, or utilized in diagnostic imaging when attachedto a radioactively labeled monoclonal antibody which recognizesactivated endothelial cells.

FORMULATION AND ADMINISTRATION

[0215] As noted above, anti-angiogenic compositions of the presentinvention may be formulated in a variety of forms (e.g., microspheres,pastes, films or sprays). Further, the compositions of the presentinvention may be formulated to contain more than one anti-angiogenicfactor, to contain a variety of additional compounds, to have certainphysical properties (e.g., elasticity, a particular melting point, or aspecified release rate). Within certain embodiments of the invention,compositions may be combined in order to achieve a desired effect (e.g.,several preparations of microspheres may be combined in order to achieveboth a quick and a slow or prolonged release of one or moreanti-angiogenic factor).

[0216] Anti-angiogenic factors and compositions of the present inventionmay be administered either alone, or in combination withpharmaceutically or physiologically acceptable carrier, excipients ordiluents. Generally, such carriers should be nontoxic to recipients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDT,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplarappropriate diluents.

[0217] As noted above, anti-angiogenic factors, anti-angiogeniccompositions, or pharmaceutical compositions provided herein may beprepared for administration by a variety of different routes, includingfor example intrarticularly, intraocularly, intranasally, intradermally,sublingually, orally, topically, intravesically, intrathecally,topically, intravenously, intraperitoneally, intracranially,intramuscularly, subcutaneously, or even directly into a tumor ordisease site. Other representative routes of administration includegastroscopy, ECRP and colonoscopy, which do not require full operatingprocedures and hospitalization, but may require the presence of medicalpersonnel.

[0218] The anti-angiogenic factors, anti-angiogenic compositions andpharmaceutical compositions provided herein may be placed withincontainers, along with packaging material which provides instructionsregarding the use of such materials. Generally, such instructions willinclude a tangible expression describing the reagent concentration, aswell as within certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) which may benecessary to reconstitute the anti-angiogenic factor, anti-angiogeniccomposition, or pharmaceutical composition.

[0219] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1 Preparation of Anti-Invasive Factor

[0220] The shoulder girdle and skull from a dogfish is excised, thenscraped with a scalpel in order to remove all muscle and associatedconnective tissue from the cartilage. The cartilage is then homogenizedwith a tissue grinder, and extracted by continuous stirring at roomtemperature for 2 to 5 days in a solution containing 2.0 M guanidiumhydrochloride and 0.02 M MES at pH 6.0

[0221] After 2 to 5 days, the cartilage extract is passed through gauzenetting in order to remove the larger constituents. The filtrate is thenpassed through an Amicon ultrafiltration unit which utilizesspiral-wound cartridges, with a molecular weight cutoff of 100,000. Thefiltrate (containing proteins with a molecular weight of less than100,000 daltons) is then dialyzed against 0 02 M VIES buffer (pH 6) withan Amicon ultrafiltration unit which retains proteins with a molecularweight of greater than 3,000 daltons. Utilizing this method, lowmolecular weight proteins and constituents are removed, as well asexcessive amounts of guanidium HCl. The dialysate is concentrated to afinal concentration 9 mg/ml.

Example 2 Analysis of Various Agents for Anti-Angiogenic Activity

[0222] A. Chick Chorioallantoic Membrane (“Cam”) Assays

[0223] Fertilized, domestic chick embryos were incubated for 3 daysprior to shell-less culturing. In this procedure, the egg contents wereemptied by removing the shell located around the air space. The interiorshell membrane was then severed and the opposite end of the shell wasperforated to allow the contents of the egg to gently slide out from theblunted end. The egg contents were emptied into round-bottom sterilizedglass bowls and covered with petri dish covers. These were then placedinto an incubator at 90% relative humidity and 3% CO, and incubated for3 days.

[0224] Paclitaxel (Sigma, St. Louis, Mo.) was mixed at concentrations of1, 5, 10, 30 μg per 10 ml aliquot of 0.5% aqueous methylcellulose. Sincepaclitaxel is insoluble in water, glass beads were used to produce fineparticles. Ten microliter aliquots of this solution were dried onparafilm for 1 hour forming disks 2 mm in diameter. The dried diskscontaining paclitaxel were then carefully placed at the growing edge ofeach CAM at day 6 of incubation. Controls were obtained by placingpaclitaxel-free methylcellulose disks on the CAMs over the same timecourse. After a 2 day exposure (day 8 of incubation) the vasculature wasexamined with the aid of a stereomicroscope. Liposyn II, a white opaquesolution, was injected into the CAM to increase the visibility of thevascular details. The vasculature of unstained, living embryos wereimaged using a Zeiss stereomicroscope which was interfaced with a videocamera (Dage-MTI Inc., Michigan City, Ind.). These video signals werethen displayed at 160 times magnification and captured using an imageanalysis system (Vidas, Kontron; Etching, Germany). Image negatives werethen made on a graphics recorder (Model 3000; Matrix Instruments,Orangeburg, N.Y.).

[0225] The membranes of the 8 day-old shell-less embryo were floodedwith 2% glutaraldehyde in 0.1M Na cacodylate buffer; additional fixativewas injected under the CAM. After 10 minutes in situ, the CAM wasremoved and placed into fresh fixative form hours at room temperature.The tissue was then washed overnight in cacodylate buffer containing 6%sucrose. The areas of interest were postfixed in 1% osmium tetroxide for1.5 hours at 4° C. The tissues were then dehydrated in a graded seriesof ethanols, solvent exchanged with propylene oxide, and embedded inSpurr resin. Thin sections were cut with a diamond knife, placed oncopper grids. stained, and examined in a Joel 1200EX electronmicroscope. Similarly, 0.5 mm sections were cut and stained with tolueneblue for light microscopy.

[0226] At day 11 of development, chick embryos were used for thecorrosion casting technique. Mercox resin (Ted Pella, Inc., Redding,Calif.) was injected into the CAM vasculature using a 30-gaugehypodermic needle. The casting material consisted of 2.5 grams of MercoxCL-2B polymer and 0.05 grams of catalyst (55% benzoyl peroxide) having a5 minute polymerization time. After injection, the plastic was allowedto sit in situ for an hour at room temperature and then overnight in anoven at 65° C. The CAM it was then placed in 50% aqueous solution ofsodium hydroxide to digest all organic components. The plastic castswere washed extensively in distilled water, air-dried, coated withgold/palladium, and viewed with the Philips 501B scanning electronmicroscope.

[0227] Results of the above experiments are shown in FIGS. 1-4. Briefly,the general features of the normal chick shell-less egg culture areshown in FIG. 1A. At day 6 of incubation, the embryo is centrallypositioned to a radially expanding network of blood vessels; the CAMdevelops adjacent to the embryo. These growing vessels lie close to thesurface and are readily visible making this system an idealized modelfor the study of angiogenesis. Living, unstained capillary networks ofthe CAM can be imaged noninvasively with a stereomicroscope. FIG. 1Billustrates such a vascular area in which the cellular blood elementswithin capillaries were recorded with the use of a video/computerinterface. The 3-dimensional architecture of such CAM capillary networksis shown by the corrosion casting method and viewed in the scanningelectron microscope (FIG. 1C). These castings revealed underlyingvessels which project toward the CAM surface where they form a singlelayer of anastomotic capillaries.

[0228] Transverse sections through the CAM show an outer ectodermconsisting of a double cell layer, a broader mesodermal layer containingcapillaries which lie subjacent to the ectoderm, adventitial cells, andan inner, single endodermal cell layer (FIG. 1D). At the electronmicroscopic level, the typical structural details of the CAM capillariesare demonstrated. Typically, these vessels lie in close association withthe inner cell layer of ectoderm (FIG. 1E)

[0229] After 48 hours exposure to paclitaxel at concentrations of 0.25,0.5, 1, 5, 10, or 30 ug, each CAM was examined under living conditionswith a stereomicroscope equipped with a video/computer interface inorder to evaluate the effects on angiogenesis. This imaging setup wasused at a magnification of 160 times which permitted the directvisualization of blood cells within the capillaries; thereby blood flowin areas of interest could be easily assessed and recorded. For thisstudy, the inhibition of angiogenesis was defined as an area of the CAMlacking a capillary network and vascular blood flow. Throughout theexperiments, avascular zones were assessed on a 4 point avasculargradient (Table I). This scale represents the degree of overallinhibition with maximal inhibition represented as a 3 on the avasculargradient scale. Paclitaxel was very consistent and induced a maximalavascular zone (6 mm in diameter or a 3 on the avasculare gradientscale) within 48 hours depending on its concentration. TABLE I AVASCULARGRADIENT 0 normal vascularity 1 lacking some microvascular movement 2*small avascular zone approximately 2 mm in diameter 3* avascularityextending beyond the disk (6 mm in diameter)

[0230] The dose-dependent, experimental data of the effects ofpaclitaxel at different concentrations are shown in Table II. TABLE IIAngiogenic Inhibition by Paclitaxel Paclitaxel in Methylcellulose DisksEmbryos Evaluated (μg) positive/total) % Inhibition 0.25 2/11 18 0.56/11 54 1 6/15 40 5 20/27 76 10 16/21 76 30 31/31 100 0 (control) 0/40 0

[0231] TABLE III Angiogenic Inhibition of Paclitaxel-Loaded ThermopastePaclitaxel-loaded Thermopaste (%) Embryos Evaluated (positive/n) 0.25414 0.5 4/4 1 8/8 5 4/4 10 5/5 20 6/6 0 (control)  0/30

[0232] Typical paclitaxel-treated CAMs are also shown with thetransparent methylcellulose disk centrally positioned over the avascularzone measuring 6 mm in diameter. At a slightly higher magnification, theperiphery of such avascular zones is clearly evident (FIG. 2C); thesurrounding functional vessels were often redirected away from thesource of paclitaxel (FIGS. 2C and 2D). Such angular redirecting ofblood flow was never observed under normal conditions. Another featureof the effects of paclitaxel was the formation of blood islands withinthe avascular zone representing the aggregation of blood cells.

[0233] The associated morphological alterations of thepaclitaxel-treated CAM are readily apparent at both the light andelectron microscopic levels. For the convenience of presentation, threedistinct phases of general transition from the normal to the avascularstate are shown. Near the periphery of the avascular zone the CAM ishallmarked by an abundance of mitotic cells within all three germ layers(FIGS. 3A and 4A). This enhanced mitotic division was also a consistentobservation for capillary endothelial cells. However, the endothelialcells remained junctionally intact with no extravasation of blood cells.With further degradation, the CAM is characterized by the breakdown anddissolution of capillaries (FIGS. 3B and 4B). The presumptiveendothelial cells, typically arrested in mitosis, still maintain a closespatial relationship with blood cells and lie subjacent to the ectoderm;however, these cells are not junctionally linked. The most centralportion of the avascular zone was characterized by a thickenedectodermal and endodermal layer (FIGS. 3C and 4C). Although these layerswere thickened, the cellular junctions remained intact and the layersmaintained their structural characteristics. Within the mesoderm,scattered mitotically arrested cells were abundant: these cells did notexhibit the endothelial cell polarization observed in the former phase.Also, throughout this avascular region, degenerating cells were commonas noted by the electron dense vacuoles and cellular debris (FIG. 4C).

[0234] In summary, this study demonstrated that 48 hours afterpaclitaxel application to the CAM, angiogenesis was inhibited. The bloodvessel inhibition formed an avascular zone which was represented bythree transitional phases of paclitaxel's effect. The central, mostaffected area of the avascular zone contained disrupted capillaries withextravasated red blood cells; this indicated that intercellularjunctions between endothelial cells were absent. The cells of theendoderm and ectoderm maintained their intercellular junctions andtherefore these germ layers remained intact; however, they were slightlythickened. As the normal vascular area was approached, the blood vesselsretained their junctional complexes and therefore also remained intact.At the periphery of the paclitaxel-treated zone, further blood vesselgrowth was inhibited which was evident by the typical redirecting or“elbowing” effect of the blood vessels (FIG. 2D).

[0235] Paclitaxel-treated avascular zones also revealed an abundance ofcells arrested in mitosis in all three germ layers of the CAM; this wasunique to paclitaxel since no previous study has illustrated such anevent. By being arrested in mitosis, endothelial cells could not undergotheir normal metabolic functions involved in angiogenesis. Incomparison, the avascular zone formed by suramin and cortisone acetatedo not produce mitotically arrested cells in the CAM; they onlyprevented further blood vessel growth into the treated area. Therefore,even though these agents are anti-angiogenic, there are many points inwhich the angiogenesis process may be targeted.

[0236] The effects of paclitaxel over the 48 hour duration were alsoobserved. During this period of observation it was noticed thatinhibition of angiogenesis occurs as early as 9 hours after application.Histological sections revealed a similar morphology as seen in the firsttransition phase of the avascular zone at 48 hours illustrated in FIGS.3A and 4A. Also, we observed in the revascularization process into theavascular zone previously observed. It has been found that the avascularzone formed by heparin and angiostatic steroids became revascularized 60hours after application. In one study, paclitaxel-treated avascularzones did not revascularize for at least 7 days after applicationimplying a more potent long-term effect.

Example 3 Encapsulation of Suramin

[0237] One milliliter of 5% ELVAX (poly(ethylene-vinyl acetate)cross-linked with 5% vinyl acetate) in dichloromethane (“DCM”) is mixedwith a fixed weight of sub-micron ground sodium suramin. This mixture isinjected into 5 ml of 5% Polyvinyl Alcohol (“PVA”) in water in a 30 mlflat bottomed test tube. Tubes containing different weights of the drugare then suspended in a multi-sample water bath at 40° for 90 minuteswith automated stirring. The mixtures are removed, and microspheresamples taken for size analysis. Tubes are centrifuged at 1000 g for 5min. The PVA supernatant is removed and saved for analysis(nonencapsulated drug). The microspheres are then washed (vortexed) in 5ml of water and recentrifuged. The 5 ml wash is saved for analysis(surface bound drug). Microspheres are then wetted in 50 ul of methanol,and vortexed in 1 ml of DCM to dissolve the ELVAX. The microspheres arethen warmed to 40° C., and 5 ml of 50° C. water is slowly added withstirring. This procedure results in the immediate evaporation of DCM,thereby causing the release of sodium suramin into the 5 ml of water.

[0238] All samples were assayed for drug content by quantification offluorescence. Briefly, sodium suramin absorbs uv/vis with a lambda maxof 312 nm. This absorption is linear in the 0 to 100 ug/ml range in bothwater and 5% PVA. Sodium suramin also fluoresces strongly with anexcitation maximum at 312 nm, and emission maximum at 400 nm. Thisfluorescence is quantifiable in the 0 to 25 ug/ml range.

[0239] The results of these experiments is shown in FIGS. 5-11. Resultsare shown in FIGS. 5-10. Briefly, the size distribution of microspheresby number (FIG. 5) or by weight (FIG. 6) appears to be unaffected byinclusion of the drug in the DCM. Good yields of microspheres in the 20to 60 μm range may be obtained.

[0240] The encapsulation of suramin is very low (<1%) (see FIG. 8).However as the weight of drug is increased in the DCM the total amountof drug encapsulated increased although the % encapsulation decreased.As is shown in FIG. 7, 50 ug of drug may be encapsulated in 50 mg ofELVAX. Encapsulation of sodium suramin in 2.5% PVA containing 10% NaClis shown in FIG. 9 (size distribution by weight). Encapsulation ofsodium suramin in 5% PVA containing 10% NaCl is shown in FIGS. 10 and 11(size distribution by weight, and number, respectively).

[0241] To assess suramin and cortisone acetate as potentialanti-angiogenic agents, each agent was mixed with 0.5% methylcelluloseand applied the dried disks containing the agent onto the developingblood vessels of the 6-day old CAM. A combination treatment of suramin(70 μg) with cortisone acetate (20 μg) was successful in inhibitingangiogenesis when tested on the CAM for 48 hours. The resultingavascular region measured 6 mm in diameter and revealed an absence ofblood flow and the appearance of sparse blood islands (FIGS. 28A and28B).

Example 4 Encapsulation of Paclitaxel

[0242] Five hundred micrograms of either paclitaxel or baccatin (apaclitaxel analog, available from Inflazyme Pharmaceuticals Inc.,Vancouver, British Columbia, Canada) are dissolved in 1 ml of a 50-50ELVAX:poly-1-lactic acid mixture in dcm. Microspheres are then preparedin a dissolution machine (Six-spindle dissolution tester, VanderKanp,Van Kell Industries Inc., U.S.A.) in triplicate at 200 rpm, 42° C., for3 hours. Microspheres so prepared are washed twice in water and sized onthe microscope.

[0243] Determination of paclitaxel encapsulation is undertaken in auv/vis assay (uv/vis lambda max. at 237 nm, fluorescence assay atexcitation 237, emission at 325 nm; Fluorescence results are presentedin square brackets []). Utilizing the procedures described above, 58 μg(+/−12 μg) [75 μg (+/−25 μg)] of paclitaxel may be encapsulated from atotal 500 μg of starting material. This represents 12% (+/−2.4%) [15%(+/−5%)] of the original weight, or 1.2% (+/−0.25%) [1.5% (+/−0.5%)] byweight of the polymer. After 18 hours of tumbling in an oven at 37° C.,10.3% (+/−10%) [6% (+/−5.6%)] of the total paclitaxel had been releasedfrom the microspheres..

[0244] For baccatin, 100+/−15 μg [83+/−231 g] of baccatin can beencapsulated from a total of 500 μg starting material. This represents a20% (+/−3%) [17% (+/−5%) of the original weight of baccatin, and2%(+/−0.3%) [1.7% (+/−0.5%)] by weight of the polymer. After 18 hours oftumbling in an oven at 37° C., 55% (+/−13%) [60% (+/−23%)] of thebaccatin is released from the microspheres.

Example 5 Analysis of Surgical Paste Containing Anti-AngiogenicCompositions

[0245] Fisher rats weighing approximately 300 grams are anesthetized,and a 1 cm transverse upper abdominal incision is made. Two-tenths of amilliliter of saline containing 1×10⁶ live 9L gliosarcoma cells (elutedimmediately prior to use from tissue culture) are injected into 2 of the5 hepatic lobes by piercing a 27 gauge needle 1 cm through the livercapsule. The abdominal wound is closed with 6.0 resorptible suture andskin clips and the GA terminated.

[0246] After 2 weeks, the tumor deposits will measure approximately 1cm. At this time, both hepatic tumors are resected and the bare marginof the liver is packed with a hemostatic agent. The rats are dividedinto two groups: half is administered polymeric carrier alone, and theother half receives an anti-angiogenic composition.

[0247] Rats are sacrificed 2, 7, 14, 21 and 84 days post hepaticresection. In particular, the rats are euthanized by injecting Euthanylinto the dorsal vein of the tail. The liver, spleen, and both lungs areremoved, and histologic analysis is performed in order to study thetumors for evidence of anti-angiogenic activity.

Example 6 Embolization of Rat Arteries

[0248] Fisher rats weighing approximately 300 grams are anesthetized.Utilizing aseptic procedures, a 1 cm transverse upper abdominal incisionis made, and the liver identified. Two-tenths of a milliliter of salinecontaining 1 million live 9L gliosarcoma cells (eluted immediately priorfrom tissue culture) is injected into each of the 5 hepatic lobes bypiercing a 27 gauge needle 1 cm through the liver capsule. One-tenth ofa milliliter of normal saline is injected into the needle as it iswithdrawn to ensure that there is no spillage of cells into theperitoneal cavity. A pledget of gelfoam is placed on each of thepuncture sites to ensure hemostasis. The abdominal wound is closed with6.0 resorptible suture with skin clips, and the anesthetic terminated.The rat is returned to the animal care facility to have a standard dietfor 14 days, at which time each tumor deposit will measure 1 cm indiameter. The same procedure is repeated using Westar rats and a ColonCancer cell line (Radiologic Oncology Lab, M. D. Anderson, Houston,Tex.). In this instance, 3 weeks are required post-injection for thetumor deposits to measure 1 cm in diameter each.

[0249] After 2 or 3 weeks, depending on the rat species, the samegeneral anesthetic procedure is followed and a midline abdominalincision is performed. The duodenum is flipped and the gastroduodenalartery is identified and mobilized. Ties are placed above and below acutdown site on the midportion of the gastroduodenal artery (GDA), and0.038 inch polyethylene tubing is introduced in a retrograde fashioninto the artery using an operating microscope. The tie below theinsertion point will ligate the artery, while the one above will fix thecatheter in place. Angiography is performed by injecting 0.5 ml of 60%radiopaque contrast material through the catheter as an x-ray is taken.The hepatic artery is then embolized by refluxing particles measuring15-200 μm through the gastroduodenal artery catheter until flow,observed via the operating microscope, is seen to cease for at least 30seconds. Occlusion of the hepatic artery is confirmed by repeating anangiogram through the GDA catheter. Utilizing this procedure, one-halfof the rats receive 15-200 μm particles of polymer alone, and the otherhalf receive 15-200 μm particles of the polymer-anti-angiogenic factorcomposition. The upper GDA ligature is tightened to occlude the GDA asthe catheter is withdrawn to ensure hemostasis, and the hepatic artery(although embolized) is left intact. The abdomen is closed with 6.0absorbable suture and surgical clips.

[0250] The rats are subsequently sacrificed at 2, 7, 14, 21 and 84 dayspost-embolization in order to determine efficacy of the anti-angiogenicfactor. Briefly, general anesthetic is given, and utilizing asepticprecautions, a midline incision performed. The GDA is mobilized again,and after placing a ligature near the junction of the GDA and thehepatic artery (i.e., well above the site of the previous cutdown), a0.038-inch polyethylene tubing is inserted via cutdown of the vessel andangiography is performed. The rat is then euthanized by injectingEuthanyl into the dorsal vein of the tail. Once euthanasia is confirmed,the liver is removed en bloc along with the stomach, spleen and bothlungs.

[0251] Histologic analysis is performed on a prepared slide stained withhematoxylin and eosin (“H and E”) stain. Briefly, the lungs aresectioned at 1 cm intervals to assess passage of embolic materialthrough the hepatic veins and into the right side of circulation. Thestomach and spleen are also sectioned in order to assess inadvertentimmobilization from reflux of particles into the celiac access of thecollateral circulation.

Example 7 Transplantation of Biliary Stents in Rats

[0252] General anesthetic is administered to 300 gram Fisher rats. A 1cm transverse incision is then made in the upper abdomen, and the liveridentified. In the most superficial lobe, 0.2 ml of saline containing 1million cells of 9L gliosarcoma cells (eluted from tissue cultureimmediately prior to use) is injected via a 27 gauge needle to a depthof 1 cm into the liver capsule. Hemostasis is achieved after removal ofthe needle by placing a pledget of gelfoam at the puncture sites. Salineis injected as the needle is removed to ensure no spillage of cells intothe peritoneal cavity or along the needle track. The general anestheticis terminated, and the animal returned to the animal care center andplaced on a normal diet.

[0253] Two weeks later, general anesthetic is administered, andutilizing aseptic precautions, the hepatic lobe containing the tumor isidentified through a midline incision. A 16 gauge angiographic needle isthen inserted through the hepatic capsule into the tumor, a 0.038-inchguidewire passed through the needle, and the needle withdrawn over theguidewire. A number 5 French dilator is passed over the guide into thetumor and withdrawn. A number 5 French delivery catheter is then passedover the wire containing a self-expanding stainless steel Wallstent (5mm in diameter and 1 cm long). The stent is deployed into the tumor andthe guidewire delivery catheter is removed. One-third of the rats have aconventional stainless steel stent inserted into the tumor, one-third astainless steel stent coated with polymer, and one third a stent coatedwith the polymer-anti-angiogenic factor compound. The general anestheticis terminated and the rat returned to the animal care facility.

[0254] A plain abdominal X-ray is performed at 2 days in order to assessthe degree of stent opening. Rats are sacrificed at 2, 7, 14, 28 and 56days post-stent insertion by injecting Euthanyl, and their liversremoved en bloc once euthanasia is confirmed. After fixation informaldehyde for 48 hours, the liver is sectioned at 0.5 mm intervals;including severing the stent transversely, using a fresh blade for eachslice. Histologic sections stained with H and E are then analyzed toassess the degree of tumor ingrowth into the stent lumen.

Example 8 Manufacture of Microspheres

[0255] Equipment which is preferred for the manufacture of microspheresdescribed below include: 200 ml water jacketed beaker (Kimax or Pyrex),Haake circulating water bath, overhead stirrer and controller with 2inch diameter (4 blade, propeller type stainless steel stirrer - Fisherbrand), 500 ml glass beaker, hot plate/stirrer (Coming brand), 4×50 mlpolypropylene centrifuge tubes (Nalgene), glass scintillation vials withplastic insert caps, table top centrifuge (GPR Beckman), high speedcentrifuge- floor model (JS 21 Beckman), Mettler analytical balance (AJ100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg),automatic pipetter (Gilson). Reagents include Polycaprolactone(“PCL”—mol wt 10,000 to 20,000; Polysciences, Warrington Pa., USA).“washed” (see later method of “washing”) Ethylene Vinyl Acetate (“EVA”),Poly(DL)lactic acid (“PLA”—mol wt 15,000 to 25,000; Polysciences),Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000: 99% hydrolyzed;Aldrich Chemical Co.. Milwaukee Wis. USA), Dichloromethane (“DCM” or“methylene chloride”; HPLC grade Fisher scientific), and distilledwater.

[0256] A. Preparation of 5% (w/v) Polymer Solutions

[0257] Depending on the polymer solution being prepared. 1.00 g of PCLor PLA, or 0.50 g each of PLA and washed EVA is weighed directly into a20 ml glass scintillation vial. Twenty milliliters of DCM is then added,and the vial tightly capped. The vial is stored at room temperature (25°C.) for one hour (occasional shaking may be used), or until all thepolymer has dissolved (the solution should be clear). The solution maybe stored at room temperature for at least two weeks.

[0258] B. Preparation of 5% (w/v) Stock Solution of PVA

[0259] Twenty-five grams of PVA is weighed directlv into a 600 ml glassbeaker. Five hundred milliliters of distilled water is added, alone witha 3 inch Teflon coated stir bar. The beaker is covered with class todecrease evaporation losses, and placed into a 2000 ml glass beakercontaining 300 ml of water (which acts as a water bath). The PVA isstirred at 300 rpm at 85° C. (Corning hot plate/stirrer) for 2 hours oruntil fully dissolved. Dissolution of the PVA may be determined by avisual check; the solution should be clear. The solution is thentransferred to a glass screw top storage container and stored at 4° C.for a maximum of two months. The solution, however should be warmed toroom temperature before use or dilution.

[0260] C. Procedure for Producing Microspheres

[0261] Based on the size of microspheres being made (see Table 1), 100ml of the PVA solution (concentrations given in Table IV) is placed intothe 200 ml water jacketed beaker. Haake circulating water bath isconnected to this beaker and the contents are allowed to equilibrate at27° C. (+/−10° C.) for 10 minutes. Based on the size of microspheresbeing made (see Table IV), the start speed of the overhead stirrer isset, and the blade of the overhead stirrer placed half way down in thePVA solution. The stirrer is then started, and 10 ml of polymer solution(polymer solution used based on type of microspheres being produced) isthen dripped into the stirring PVA over a period of 2 minutes using a 5ml automatic pipetter. After 3 minutes the stir speed is adjusted (seeTable IV), and the solution stirred for an additional 2.5 hours. Thestirring blade is then removed from the microsphere preparation, andrinsed with 10 ml of distilled water so that the rinse solution drainsinto the microsphere preparation. The microsphere preparation is thenpoured into a 500 ml beaker, and the jacketed water bath washed with 70ml of distilled water, which is also allowed to drain into themicrosphere preparation. The 180 ml microsphere preparation is thenstirred with a glass rod, and equal amounts are poured into fourpolypropylene 50 ml centrifuge tubes. The tubes are then capped, andcentrifuged for 10 minutes (force given in Table III). A 5 ml automaticpipetter or vacuum suction is then utilized to draw 45 ml of the PVAsolution off of each microsphere pellet. TABLE III PVA concentrations,stir speeds, and centrifugal force requirements for each diameter rangeof microspheres. PRODUC- MICROSPHERE DIAMETER RANGES TION STAGE 30 μm to100 μm 10 μm to 30 μm 0.1 μm to 3 μm PVA 2.5% (w/v) (i.e.,) 5% (w/v)(i.e., 3.5% (w/v) (i.e., concentration dilute 5% stock undiluted stock)dilute 5% stock with distilled with distilled water water Starting Stir500 rpm 500 rpm 3000 rpm Speed +/− 50 rpm +/− 50 rpm +/− 200 rpmAdjusted Stir 500 rpm 500 rpm 2500 rpm Speed +/− 50 rpm +/− 50 rpm +/−200 rpm Centrifuge 1000 g 1000 g 10 000 g Force +/− 100 g +/− 100 g +/−1000 g (Table top model) (Table top model) (High speed model)

[0262] Five milliliters of distilled water is then added to eachcentrifuge tube, which is then vortexed to resuspend the microspheres.The four microsphere suspensions are then pooled into one centrifugetube alone with 20 ml of distilled water, and centrifuged for another 10minutes (force given in Table 1). This process is repeated twoadditional times for a total of three washes. The microspheres are thencentrifuged a final time, and resuspended in 10 ml of distilled water.After the final wash, the microsphere preparation is transferred into apreweighed glass scintillation vial. The vial is capped, and leftovernight at room temperature (25° C.) in order to allow themicrospheres to sediment out under gravity. Microspheres which fall inthe size range of 0.1 um to 3 um do not sediment out under gravity, sothey are left in the 10 ml suspension.

[0263] D. Drying of 10 μm to 30 μm or 30 μm to 100 μm DiameterMicrospheres

[0264] After the microspheres have sat at room temperature overnight, a5 ml automatic pipetter or vacuum suction is used to draw thesupernatant off of the sedimented microspheres. The microspheres areallowed to dry in the uncapped vial in a drawer for a period of one weekor until they are fully dry (vial at constant weight). Faster drying maybe accomplished by leaving the uncapped vial under a slow stream ofnitrogen gas (flow approx. 10 ml/min.) in the fume hood. When fully dry(vial at constant weight), the vial is weighed and capped. The labeled,capped vial is stored at room temperature in a drawer. Microspheres arenormally stored no longer than 3 months.

[0265] E. Drying of 0.1 μm to 3 μm Diameter Microspheres

[0266] This size range of microspheres will not sediment out, so theyare left in suspension at 4° C. for a maximum of four weeks. Todetermine the concentration of microspheres in the 10 ml suspension, a200 μl sample of the suspension is pipetted into a 1.5 ml preweighedmicrofuge tube. The tube is then centrifuged at 10,000 g (Eppendorftable top microfuge), the supernatant removed, and the tube allowed todry at 50° C. overnight. The tube is then reweighed in order todetermine the weight of dried microspheres within the tube.

[0267] F. Manufacture of Paclitaxel Loaded Microsphere

[0268] In order to prepare paclitaxel containing microspheres, anappropriate amount of weighed paclitaxel (based upon the percentage ofpaclitaxel to be encapsulated) is placed directly into a 20 ml glassscintillation vial. Ten milliliters of an appropriate polymer solutionis then added to the vial containing the paclitaxel, which is thenvortexed until the paclitaxel has dissolved.

[0269] Microspheres containing paclitaxel may then be producedessentially as described above in steps (C) through (E).

Example 9 Manufacture of Stent Coating

[0270] Reagents and equipment which are utilized within the followingexperiments include (medical grade stents obtained commercially from avariety of manufacturers; e.g., the “Strecker” stent) and holdingapparatus, 20 ml glass scintillation vial with cap (plastic inserttype), TLC atomizer, Nitrogen gas tank, glass test tubes (various sizesfrom 1 ml and up), glass beakers (various sizes), Pasteur pipette,tweezers, Polycaprolactone (“PCL”—mol wt 10,000 to 20,000.Polysciences), Paclitaxel (Sigma Chemical Co., St. Louis. Mo., 95%purity), Ethylene vinyl acetate (“EVA”—washed—see previous),Poly(DL)lactic acid (“PLA”—mol wt 15,000 to 25,000; Polysciences),dichloromethane (“DCM”—FTLC grade. Fisher Scientific).

[0271] A Procedure for Sprayed Stents

[0272] The following describes a typical method using a 3 mm crimpeddiameter interleaving metal wire stent of approximately 3 cm length. Forlarger diameter stents, larger volumes of polymer/drug solution areused.

[0273] Briefly, a sufficient quantity of polymer is weighed directlyinto a 20 ml glass scintillation vial, and sufficient DCM added in orderto achieve a 2% w/v solution. The vial is then capped and mixed by handin order to dissolve the polymer. The stent is then assembled in avertical orientation, tying the stent to a retort stand with nylon.Position this stent holding apparatus 6 to 12 inches above the fume hoodfloor on a suitable support (e.g, inverted 2000 ml glass beaker) toenable horizontal spraying. Using an automatic pipette, a suitablevolume (minimum 5 ml) of the 2% polymer solution is transferred to aseparate 20 ml glass scintillation vial. An appropriate amount ofpaclitaxel is then added to the solution and dissolved by hand shaking.

[0274] To prepare for spraying, remove the cap of this vial and dip thebarrel (only) of an TLC atomizer into the polymer solution. Note thatthe reservoir of the atomizer need not be used in this procedure: the 20ml glass vial acts as a reservoir. Connect the nitrogen tank to the gasinlet of the atomizer. Gradually increase the pressure until atomizationand spraying begins. Note the pressure and use this pressure throughoutthe procedure. To spray the stent use 5 second oscillating sprays with a15 second dry time between sprays. After 5 sprays, rotate the stent 90and spray that portion of the stent. Repeat until all sides of the stenthave been sprayed. During the dry time, finger crimp the gas line toavoid wastage of the spray. Spraying is continued until a suitableamount of polymer is deposited on the stents. The amount may be based onthe specific stent application in vivo. To determine the amount, weighthe stent after spraying has been completed and the stent has dried.Subtract the original weight of the stent from the finished weight andthis produces the amount of polymer (plus paclitaxel) applied to thestent. Store the coated stent in a sealed container.

[0275] B. Procedure for Dipped Stents

[0276] The following describes a typical method using a 3 mm crimpeddiameter interleaving metal wire stent of approximately 3 cm length. Forlarger diameter stents, larger volumes of polymer/drug solution are usedin larger sized test tubes.

[0277] Weigh 2 g of EVA into a 20 ml glass scintillation vial and add 20ml of DCM. Cap the vial and leave it for 2 hours to dissolve (hand shakethe vial frequently to assist the dissolving process). Weigh a knownweight of paclitaxel directly into a 1 ml glass test tube and add 0.5 mlof the polymer solution. Using a glass Pasteur pipette, dissolve thepaclitaxel by gently pumping the polymer solution. Once the paclitaxelis dissolved, hold the test tube in a near horizontal position (thesticky polymer solution will not flow out). Using tweezers, insert thestent into the tube all the way to the bottom. Allow the polymersolution to flow almost to the mouth of the test tube by angling themouth below horizontal and then restoring the test tube to an angleslightly above the horizontal. While slowly rotating the stent in thetube, slowly remove the stent (approximately 30 seconds).

[0278] Hold the stent in a vertical position to dry. Some of the sealedperforations may pop so that a hole exists in the continuous sheet ofpolymer. This may be remedied by repeating the previous dippingprocedure, however repetition of the procedure can also lead to furtherpopping and a general uneven build up of polymer. Generally, it isbetter to dip the stent just once and to cut out a section of stent thathas no popped perforations. Store the dipped stent in a sealedcontainer.

Example 10 Manufacture of Surgical “Pastes”

[0279] As noted above, the present invention provides a variety ofpolymeric-containing drug compositions that may be utilized within avariety of clinical situations. For example, compositions may beproduced: (1) as a “thermopaste” that is applied to a desired site as afluid, and hardens to a solid of the desired shape at a specifiedtemperature (e.g., body temperature); (2) as a spray (i.e., “nanospray”)which may delivered to a desired site either directly or through aspecialized apparatus (e.g., endoscopy), and which subsequently hardensto a solid which adheres to the tissue to which it is applied; (3) as anadherent, pliable, resilient, angiogenesis inhibitor-polymer filmapplied to a desired site either directly or through a specializedapparatus, and which preferably adheres to the site to which it isapplied; and (4) as a fluid composed of a suspension of microspheres inan appropriate carrier medium, which is applied to a desired site eitherdirectly or via a specialized apparatus, and which leaves a layer ofmicrospheres at the application site. Representative examples of each ofthe above embodiments is set forth in more detail below.

[0280] A. Procedure for Producing Thermopaste

[0281] Reagents and equipment which are utilized within the followingexperiments include a sterile glass syringe (1 ml), Corning hotplate/stirrer, 20 ml lass scintillation vial, moulds (e.g., 50 μl DSCpan or 50 ml centrifuge tube cap inner portion), scalpel and tweezers,Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), and Paclitaxel (Sigma grade 95% purity minimum).

[0282] Weigh 5 00 g of polycaprolactone directlv into a 20 ml glassscintillation vial. Place the vial in a 600 ml beaker containing 50 mlof water. Gently heat the beaker to 65° C. and hold it at thattemperature for 20 minutes. This allows the polymer to melt. Thoroughlymix a known weight of paclitaxel, or other angiogenesis inhibitor intothe melted polymer at 65° C. Pour the melted polymer into a prewarmed(60° C. oven) mould. Use a spatula to assist with the pouring process.Allow the mould to cool so the polymer solidifies. Cut or break thepolymer into small pieces (approximately 2 mm by 2 mm in size). Thesepieces must fit into a 1 ml glass syringe. Remove the plunger from the Iml glass syringe (do not remove the cap from the tip) and place it on abalance. Zero the balance.

[0283] Weigh 0 5 g of the pieces directly into the open end of thesyringe. Place the glass syringe upright (capped tip downwards) into a500 ml glass beaker containing distilled water at 65° C. (corning hotplate) so that no water enters the barrel. The polymer melts completelywithin 10 minutes in this apparatus. When the polymer pieces havemelted, remove the barrel from the water bath, hold it horizontally andremove the cap. Insert the plunger into the barrel and compress themelted polymer into a sticky mass at the tip end of the barrel. Cap thesyringe and allow it to cool to room temperature.

[0284] For application, the syringe may be reheated to 60° C. andadministered as a liquid which solidifies when cooled to bodytemperature.

[0285] B. Procedure for Producing Nanospray

[0286] Nanospray is a suspension of small microspheres in saline. If themicrospheres are very small (i.e., under 1 μm in diameter) they form acolloid so that the suspension will not sediment under gravity. As isdescribed in more detail below, a suspension of 0.1 μm to 1 μmmicroparticles may be created suitable for deposition onto tissuethrough a finger pumped aerosol. Equipment and materials which may beutilized to produce nanospray include 200 ml water jacketed beaker(Kimax or Pyrex), Haake circulating water bath, overhead stirrer andcontroller with 2 inch diameter (4 blade, propeller type stainless steelstirrer; Fisher brand), 500 ml glass beaker, hot plate/stirrer (Comingbrand), 4×50 ml polypropylene centrifuge tubes (Nalgene), glassscintillation vials with plastic insert caps, table top centrifuge(Beckman), high speed centrifuge—floor model (JS 21 Beckman), Mettleranalytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance(AE 163, 0.01 mg), automatic pipetter (Gilson), sterile pipette tips,pump action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood.Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), “washed” (see previous) Ethylene Vinyl Acetate(“EVA”), Poly(DL)lactic acid (“PLA” mol wt 15,000 to 25.000;Polysciences), Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000; 99%hydrolyzed; Aldrich Chemical Co., Milwaukee, Wis. USA), Dichloromethane(“DCM” or “methylene chloride;” HPLC grade Fisher scientific), Distilledwater, sterile saline (Becton and Dickenson or equivalent)

[0287] 1. Preparation of 5% (w/v) Polymer Solutions

[0288] Depending on the polymer solution being prepared, weigh 1.00 g ofPCL or PLA or 0 50 g each of PLA and washed EVA directlv into a 20 mllass scintillation vial. Using a measuring cylinder, add 20 ml of DCMand tightly cap the vial. Leave the vial at room temperature (25° C.)for one hour or until all the polymer has dissolved (occasional handshaking may be used). Dissolving of the polymer can be determined by avisual check; the solution should be clear. Label the vial with the nameof the solution and the date it was produced. Store the solutions atroom temperature and use within two weeks.

[0289] 2. Preparation of 3.5% (w/v) Stock Solution of PVA

[0290] The solution can be prepared by following the procedure givenbelow, or by diluting the 5% (w/v) PVA stock solution prepared forproduction of microspheres (see Example 8). Briefly, 17.5 g of PVA isweighed directlv into a 600 ml glass beaker, and 500 ml of distilledwater is added. Place a 3 inch Teflon coated stir bar in the beaker.Cover the beaker with a cover glass to reduce evaporation losses. Placethe beaker in a 2000 ml glass beaker containing 300 ml of water. Thiswill act as a water bath. Stir the PVA at 300 rpm at 85° C. (Corning hotplate/stirrer) for 2 hours or until fully dissolved. Dissolving of thePVA can be determined by a visual check; the solution should be clear.Use a pipette to transfer the solution to a glass screw top storagecontainer and store at 4° C. for a maximum of two months. This solutionshould be warmed to room temperature before use or dilution.

[0291] 3. Procedure for Producing Nanospray

[0292] Place the stirring assembly in a fume hood. Place 100 ml of the3.5% PVA solution in the 200 ml water jacketed beaker. Connect the Haakewater bath to this beaker and allow the contents to equilibrate at 27°C. (+/−1° C.) for 10 minutes. Set the start speed of the overheadstirrer at 3000 rpm (−/−200 rpm). Place the blade of the overheadstirrer half way down in the PVA solution and start the stirrer. Drip 10ml of polymer solution (polymer solution used based on type of nanospraybeing produced) into the stirring PVA over a period of 2 minutes using a5 ml automatic pipetter. After 3 minutes, adjust the stir speed to 2500rpm (+/−200 rpm) and leave the assembly for 2.5 hours. After 2.5 hours,remove the stirring blade from the nanospray preparation and rinse with10 ml of distilled water. Allow the rinse solution to go into thenanospray preparation.

[0293] Pour the microsphere preparation into a 500 ml beaker. Wash thejacketed water bath with 70 ml of distilled water. Allow the 70 ml rinsesolution to go into the microsphere preparation. Stir the 180 mlmicrosphere preparation with a glass rod and pour equal amounts of itinto four polypropylene 50 ml centrifuge tubes. Cap the tubes Centrifugethe capped tubes at 10,000 g (+/−1000 g) for 10 minutes. Using a 5 mlautomatic pipetter or vacuum suction, draw 45 ml of the PVA solution offof each microsphere pellet and discard it. Add 5 ml of distilled waterto each centrifuge tube and use a vortex to resuspend the microspheresin each tube. Using 20 ml of distilled water, pool the four microspheresuspensions into one centrifuge tube. To wash the microspheres,centrifuge the nanospray preparation for 10 minutes at 10,000 g (+/−1000g). Draw the supernatant off of the microsphere pellet. Add 40 ml ofdistilled water and use a vortex to resuspend the microspheres. Repeatthis process two more times for a total of three washes. Do a fourthwash but use only 10 ml (not 40 ml) of distilled water when resuspendingthe microspheres After the fourth wash, transfer the microspherepreparation into a preweighed glass scintillation vial.

[0294] Cap the vial and let it to sit for 1 hour at room temperature(25° C.) to allow the 2 μm and 3 μm diameter microspheres to sedimentout under gravity. After 1 hour, draw off the top 9 ml of suspensionusing a 5 ml automatic pipetter. Place the 9 ml into a sterile capped 50ml centrifuge tube. Centrifuge the suspension at 10,000 g (+/−1000 g)for 10 minutes. Discard the supernatant and resuspend the pellet in 20ml of sterile saline. Centrifuge the suspension at 10,000 g (+/−1000 g)for 10 minutes. Discard the supernatant and resuspend the pellet insterile saline. The quantity of saline used is dependent on the finalrequired suspension concentration (usually 10% w/v). Thoroughly rinsethe aerosol apparatus in sterile saline and add the nanospray suspensionto the aerosol.

[0295] C. Manufacture of Paclitaxel Loaded Nanospray

[0296] To manufacture nanospray containing paclitaxel, use Paclitaxel(Sigma grade 95% purity). To prepare the polymer drug stock solution,weigh the appropriate amount of paclitaxel directlv into a 20 ml glassscintillation vial. The appropriate amount is determined based on thepercentage of paclitaxel to be in the nanospray. For example, ifnanospray containing 5% paclitaxel was required, then the amount ofpaclitaxel weighed would be 25 mg since the amount of polymer added is10 ml of a 5% polymer in DCM solution (see next step).

[0297] Add 10 ml of the appropriate 5% polymer solution to the vialcontaining the paclitaxel. Cap the vial and vortex or hand swirl it todissolve the paclitaxel (visual check to ensure paclitaxel dissolved).Label the vial with the date it was produced. This is to be used the dayit is produced.

[0298] Follow the procedures as described above, except thatpolymer/drug (e.g., paclitaxel) stock solution is substituted for thepolymer solution.

[0299] D. Procedure for Producing Film

[0300] The term film refers to a polymer formed into one of manygeometric shapes. The film may be a thin, elastic sheet of polymer or a2 mm thick disc of polymer. This film is designed to be placed onexposed tissue so that any encapsulated drug is released from thepolymer over a long period of time at the tissue site. Films may be madeby several processes, including for example, by casting, and by spraying

[0301] In the casting technique, polymer is either melted and pouredinto a shape or dissolved in dichloromethane and poured into a shape.The polymer then either solidifies as it cools or solidifies as thesolvent evaporates, respectively. In the spraying technique, the polymeris dissolved in solvent and sprayed onto glass, as the solventevaporates the polymer solidifies on the glass. Repeated sprayingenables a build up of polymer into a film that can be peeled from thelass.

[0302] Reagents and equipment which were utilized within theseexperiments include a small beaker. Corning hot plate stirrer, castingmoulds (e.g,. 50 ml centrifuge tube caps) and mould holding apparatus,20 ml glass scintillation vial with cap (Plastic insert type), TLCatomizer, Nitrogen gas tank, Polycaprolactone (“PCL”—mol wt 10,000 to20,000; Polysciences), Paclitaxel (Sigma 95% purity), Ethanol, “washed”(see previous) Ethylene vinyl acetate (“EVA”), Poly(DL)lactic acid(“PLA”—mol wt 15,000 to 25,000; Polysciences), Dichloromethane (HPLCgrade Fisher Scientific).

[0303] 1. Procedure for Producing Films—Melt Casting

[0304] Weigh a known weight of PCL directly into a small glass beaker.Place the beaker in a larger beaker containing water (to act as a waterbath) and put it on the hot plate at 70° C. for 15 minutes or until thepolymer has fully melted. Add a known weight of drug to the meltedpolymer and stir the mixture thoroughly. To aid dispersion of the drugin the melted PCL, the drug may be suspended/dissolved in a small volume(<10% of the volume of the melted PCL) of 100% ethanol. This ethanolsuspension is then mixed into the melted polymer. Pour the meltedpolymer in mould and let it to cool. After cooling, store the film in acontainer.

[0305] 2. Procedure for Producing Films—Solvent Casting

[0306] Weigh a known weight of PCL directly into a 20 ml glassscintillation vial and add sufficient DCM to achieve a 10% w/v solution.Cap the vial and mix the solution. Add sufficient paclitaxel to thesolution to achieve the desired Final paclitaxel concentration. Use handshaking or vortexing to dissolve the paclitaxel in the solution. Let thesolution sit for one hour (to diminish the presence of air bubbles) andthen pour it slowly into a mould. The mould used is based on the shaperequired. Place the mould in the fume hood overnight. This will allowthe DCM to evaporate. Either leave the film in the mould to store it orpeel it out and store it in a sealed container.

[0307] 3. Procedure for Producing Films—Sprayed

[0308] Weigh sufficient polymer directly into a 20 ml glassscintillation vial and add sufficient DCM to achieve a 2% w/v solution.Cap the vial and mix the solution to dissolve the polymer (handshaking). Assemble the moulds in a vertical orientation in a suitablemould holding apparatus in the fume hood. Position this mould holdingapparatus 6 to 12 inches above the fume hood floor on a suitable support(e.g., inverted 2000 ml glass beaker) to enable horizontal spraying.Using an automatic pipette, transfer a suitable volume (minimum 5 ml) ofthe 2% polymer solution to a separate 20 ml glass scintillation vial.Add sufficient paclitaxel to the solution and dissolve it by handshaking the capped vial. To prepare for spraying, remove the cap of thisvial and dip the barrel (only) of an TLC atomizer into the polymersolution. Note: the reservoir of the atomizer is not used in thisprocedure—the 20 ml glass vial acts as a reservoir.

[0309] Connect the nitrogen tank to the gas inlet of the atomizer.Gradually increase the pressure until atomization and spraying begins.Note the pressure and use this pressure throughout the procedure. Tospray the moulds use 5 second oscillating sprays with a 15 second drytime between sprays. During the dry time, finger crimp the gas line toavoid wastage of the spray. Spraying is continued until a suitablethickness of polymer is deposited on the mould. The thickness is basedon the request. Leave the sprayed films attached to the moulds and storein sealed containers.

[0310] E. Procedure for Producing Nanopaste

[0311] Nanopaste is a suspension of microspheres suspended in ahydrophilic gel. Within one aspect of the invention, the gel or pastecan be smeared over tissue as a method of locating drug loadedmicrospheres close to the target tissue. Being water based, the pastewill soon become diluted with bodily fluids causing a decrease in thestickiness of the paste and a tendency of the microspheres to bedeposited on nearby tissue. A pool of microsphere encapsulated drug isthereby located close to the target tissue.

[0312] Reagents and equipment which were utilized within theseexperiments include glass beakers, Carbopol 925 (pharmaceutical grade,Goodyear Chemical Co.), distilled water, sodium hydroxide (1 M) in watersolution, sodium hydroxide solution (5 M) in water solution,microspheres in the 0.1 lm to 3 lm size range suspended in water at 20%w/v (See previous).

[0313] 1. Preparation of 5% w/v Carbopol Gel

[0314] Add a sufficient amount of carbopol to 1 M sodium hydroxide toachieve a 5% w/v solution. To dissolve the carbopol in the 1 M sodiumhydroxide, allow the mixture to sit for approximately one hour. Duringthis time period, stir the mixture using a glass rod. After one hour,take the pH of the mixture. A low pH indicates that the carbopol is notfully dissolved. The pH you want to achieve is 7.4. Use 5 M sodiumhydroxide to adjust the pH. This is accomplished by slowly adding dropsof 5 M sodium hydroxide to the mixture, stirring the mixture and takingthe pH of the mixture. It usually takes approximately one hour to adjustthe pH to 7.4. Once a pH of 7.4 is achieved, cover the gel and let itsit for 2 to 3 hours After this time period, check the pH to ensure itis still at 7.4. If it has changed, adjust back to pH 7.4 using 5 Msodium hydroxide. Allow the gel to sit for a few hours to ensure the pHis stable at 7.4. Repeat the process until the desired pH is achievedand is stable. Label the container with the name of the gel and thedate. The gel is to be used to make nanopaste within the next week.

[0315] 2. Procedure for Producing Nanopaste

[0316] Add sufficient 0.1 μm to 3 μm microspheres to water to produce a20% suspension of the microspheres. Put 8 ml of the 5% w/v carbopol gelin a glass beaker. Add 2 ml of the 20% microsphere suspension to thebeaker. Using a glass rod or a mixing spatula, stir the mixture tothoroughly disperse the microspheres throughout the gel. This usuallytakes 30 minutes. Once the microspheres are dispersed in the gel, placethe mixture in a storage jar Store the jar at 4° C. It must be usedwithin a one month period.

Example 11 Controlled Delivery of Paclitaxel from Microspheres Composedof a Blend of Ethylene-Vinyl-Acetate Copolymer and Poly(D.L LacticAcid), In Vivo Testing of the Microspheres on the Calm Assay

[0317] This example describes the preparation of paclitaxel-loadedmicrospheres composed of a blend of biodegradable poly (d,l-lactic acid)(PLA) polymer and nondegradable ethylene-vinyl acetate (EVA) copolymer.In addition, the in vitro release rate and anti-angiogenic activity ofpaclitaxel released from microspheres placed on a CALM are demonstrated.

[0318] Reagents which were utilized in these experiments includepaclitaxel, which is purchased from Sigma Chemical Co. (St. Louis, Mo.);PLA (molecular weight 15,000-25,000) and EVA (60% vinyl acetate)(purchased from Polysciences (Warrington, Pa.); polyvinyl alcohol (PVA)(molecular weight 124,000-186,000, 99% hydrolysed, purchased fromAldrich Chemical Co. (Milwaukee, Wis.)) and Dichloromethane (DCM) (HPLCgrade, obtained from Fisher Scientific Co). Distilled water is usedthroughout.

[0319] A. Preparation of Microspheres

[0320] Microspheres are prepared essentially as described in Example 8utilizing the solvent evaporation method. Briefly, 5% w/v polymersolutions in 20 mL DCM are prepared using blends of EVA:PLA between35:65 to 90:10. To 5 mL of 2.5% w/v PVA in water in a 20 mL glass vialis added 1 mL of the polymer solution dropwise with stirring. Sixsimilar vials are assembled in a six position overhead stirrer,dissolution testing apparatus (Vanderkamp) and stirred at 200 rpm. Thetemperature of the vials is increased from room temperature to 40° C.over 15 min and held at 40° C. for 2 hours. Vials are centrifuged at500×g and the microspheres washed three times in water. At some EVA:PLApolymer blends, the microsphere samples aggregated during the washingstage due to the removal of the dispersing or emulsifying agent, PVA.This aggregation effect could be analyzed semi-quantitatively sinceaggregated microspheres fused and the fused polymer mass floated on thesurface of the wash water. This surface polymer layer is discardedduring the wash treatments and the remaining, pelleted microspheres areweighed. The % aggregation is determined from

% aggregation=1−(weight of pelleted microspheres)×100 initial polymerweight

[0321] Paclitaxel loaded microspheres (0.6% w/w paclitaxel) are preparedby dissolving the paclitaxel in the 5% w/v polymer solution in DCM. Thepolymer blend used is 50:50 EVA:PLA A “large” size fraction and “small”size fraction of microspheres are produced by adding thepaclitaxel/polymer solution dropwise into 2.5% w/v PVA and 5% w/v PVA,respectively. The dispersions are stirred at 40° C. at 200 rpm for 2hours, centrifuged and washed 3 times in water as described previously.Microspheres are air dried and samples are sized using an opticalmicroscope with a stage micrometer. Over 300 microspheres are countedper sample. Control microspheres (paclitaxel absent) are prepared andsized as described previously.

[0322] B. Encapsulation efficiency

[0323] Known weights of paclitaxel-loaded microspheres are dissolved in1 mL DCM, 20 mL of 40%1/0 acetonitrile in water at 50° C. are added andvortexed until the DCM had been evaporated. The concentration ofpaclitaxel in the 40% acetonitrile is determined by HPLC using a mobilephase of water:methanol:acetonitrile (37:5:58) at a flow rate of 1mL/min (Beckman isocratic pump), a C8 reverse phase column (Beckman) andUV detection at 232 nm. To determine the recovery efficiency of thisextraction procedure, known weights of paclitaxel from 100-1000 μg aredissolved in 1 mL of DCM and subjected to the same extraction procedurein triplicate as described previously. Recoveries are always greaterthan 85% and the values of encapsulation efficiency are correctedappropriately.

[0324] C. Drug release studies

[0325] In 15 mL glass, screw capped tubes are placed 10 mL of 10 mMphosphate buffered saline (PBS), pH 7 4 and 35 mg paclitaxel-loadedmicrospheres. The tubes are tumbled at 37° C. and at given timeintervals, centrifuged at 1500×g for 5 min and the supernatant saved foranalysis. Microsphere pellets are resuspended in fresh PBS (10 mL) at37° C. and reincubated. Paclitaxel concentrations are determined byextraction into 1 mL DCM followed by evaporation to dryness under astream of nitrogen, reconstitution in 1 mL of 40% acetonitrile in waterand analysis using HPLC as previously described.

[0326] D. Scanning Electron Microscopy (SEM)

[0327] Microspheres are placed on sample holders, sputter coated withgold and micrographs obtained using a Philips 501B SEM operating at 15kV.

[0328] E. CAM Studies

[0329] Fertilized, domestic chick embryos are incubated for 4 days priorto shell-less culturing. The egg contents are incubated at 90% relativehumidity and 3% CO₂ for 2 days. On day 6 of incubation, 1 mg aliquots of0.6% paclitaxel loaded or control (paclitaxel free) microspheres areplaced directly on the CAM surface. After a 2 day exposure thevasculature is examined using a stereomicroscope interfaced with a videocamera; the video signals are then displayed on a computer and videoprinted.

[0330] F. Results

[0331] Microspheres prepared from 100% EVA are freely suspended insolutions of PVA but aggregated and coalesced or fused extensively onsubsequent washing in water to remove the PVA. Blending EVA with anincreasing proportion of PLA produced microspheres showing a decreasedtendency to aggregate and coalesce when washed in water, as described inFIG. 15A. A 50:50 blend of EVA:PLA formed microspheres with goodphysical stability, that is the microspheres remained discrete and wellsuspended with negligible aggregation and coalescence.

[0332] The size range for the “small” size fraction microspheres isdetermined to be >95% of the microsphere sample (by weight) between10-30 mm and for the “large” size fraction, >95% of the sample (byweight) between 30-100 mm. Representative scanning electron micrographsof paclitaxel loaded 50:50 EVA:PLA microspheres in the “small” and“large” size ranges are shown in FIGS. 15B and 15C, respectively, themicrospheres are spherical with a smooth surface and with no evidence ofsolid drug on the surface of the microspheres. The efficiency of loading50:50 EVA:PLA microspheres with paclitaxel is between 95-100% at initialpaclitaxel concentrations of between 100-1000 mg paclitaxel per 50 mgpolymer. There is no significant difference (Student t-test, p <0.05)between the encapsulation efficiencies for either “small” or “large”microspheres.

[0333] The time course of paclitaxel release from 0.6% w/v loaded 50:50EVA:PLA microspheres is shown in FIG. 15D for “small” size (opencircles) and “large” size (closed circles) microspheres. The releaserate studies are carried out in triplicate tubes in 3 separateexperiments. The release profiles are biphasic with an initial rapidrelease of paclitaxel or “burst” phase occurring over the first 4 daysfrom both size range microspheres. This is followed by a phase of muchslower release. There is no significant difference between the releaserates from “small” or “large” microspheres. Between 10-13% of the totalpaclitaxel content of the microspheres is released in 50 days.

[0334] The paclitaxel loaded microspheres (0.6% w/v loading) are testedusing the CAM assay and the results are shown in FIG. 15E. Thepaclitaxel microspheres released sufficient drug to produce a zone ofavascularity in the surrounding tissue (FIG. 15F). Note that immediatelyadjacent to the microspheres (“MS” in FIGS. 15E and 15F) is an area inwhich blood vessels are completely absent (Zone 1); further from themicrospheres is an area of disrupted, non-functioning capillaries (Zone2); it is only at a distance of approximately 6 mm from the microspheresthat the capillaries return to normal. In CAMs treated with controlmicrospheres (paclitaxel absent) there is a normal capillary networkarchitecture (figure not shown.)

[0335] Discussion

[0336] Arterial chemoembolization is an invasive surgical technique.Therefore, ideally, a chemoembolic formulation of an anti-angiogenicdrug such as paclitaxel would release the drug at the tumor site atconcentrations sufficient for activity for a prolonged period of time,of the order of several months. EVA is a tissue compatible nondegradablepolymer which has been used extensively for the controlled delivery ofmacromolecules over long time periods (>100 days).

[0337] EVA is initially selected as a polymeric biomaterial forpreparing microspheres with paclitaxel dispersed in the polymer matrix.However, microspheres prepared with 100% EVA aggregated and coalescedalmost completely during the washing procedure.

[0338] Polymers and copolymers based on lactic acid and glycolic acidare physiologically inert and biocompatible and degrade by hydrolysis totoxicologically acceptable products. Copolymers of lactic acid andglycolic acids have faster degradation rates than PLA and drug loadedmicrospheres prepared using these copolymers are unsuitable forprolonged, controlled release over several months. Dollinger and Sawanblended PLA with EVA and showed that the degradation lifetime of PLA isincreased as the proportion of EVA in the blend is increased. Theysuggested that blends of EVA and PLA should provide a polymer matrixwith better mechanical stability and control of drug release rates thanPLA.

[0339]FIG. 15A shows that increasing the proportion of PLA in a EVA:PLAblend decreased the extent of aggregation of the microspheresuspensions. Blends of 50% or less EVA in the EVA:PLA matrix producedphysically stable microsphere suspensions in water or PBS. A blend of50:50 EVA:PLA is selected for all subsequent studies.

[0340] Different size range fractions of microspheres could be preparedby changing the concentration of the emulsifier, PVA, in the aqueousphase. “Small” microspheres are produced at the higher PVA concentrationof 5% w/v whereas “large” microspheres are produced at 2.5% w/v PVA. Allother production variables are the same for both microsphere sizefractions. The higher concentration of emulsifier gave a more viscousaqueous dispersion medium and produced smaller droplets ofpolymer/paclitaxel/DCM emulsified in the aqueous phase and thus smallermicrospheres. The paclitaxel loaded microspheres contained between95-100% of the initial paclitaxel added to the organic phaseencapsulated within the solid microspheres. The low water solubility ofpaclitaxel favoured partitioning into the organic phase containing thepolymer.

[0341] Release rates of paclitaxel from the 50:50 EVA:PLA microspheresare very slow with less than 15% of the loaded paclitaxel being releasedin 50 days. The initial burst phase of drug release may be due todiffusion of drug from the superficial region of the microspheres (closeto the microsphere surface).

[0342] The mechanism of drug release from nondegradable polymericmatrices such as EVA is thought to involve the diffusion of waterthrough the dispersed drug phase within the polymer, dissolution of thedrug and diffusion of solute through a series of interconnecting, fluidfilled pores. Blends of EVA and PLA have been shown to be immiscible orbicontinuous over a range of 30 to 70% EVA in PLA. In degradationstudies in PBS buffer at 37° C., following an induction or lag period,PLA hydrolytically degraded and eroded from the EVA:PLA polymer blendmatrix leaving an inactive sponge-like skeleton. Although the inductionperiod and rate of PLA degradation and erosion from the blended matricesdepended on the proportion of PLA in the matrix and on process history,there is consistently little or no loss of PLA until after 40-50 days.

[0343] Although some erosion of PLA from the 50:50 EVA:PLA microspheresmay have occurred within the 50 days of the in vitro release rate study(FIG. 15C), it is likely that the primary mechanism of drug release fromthe polymer blend is diffusion of solute through a pore network in thepolymer matrix.

[0344] At the conclusion of the release rate study, the microspheres areanalyzed from the amount of drug remaining. The values for the percentof paclitaxel remaining in the 50 day incubation microsphere samples are94%+/−9% and 89% +/−12% for “large” and “small” size fractionmicrospheres, respectively.

[0345] Microspheres loaded with 6 mg per mg of polymer (0.6%) providedextensive inhibition of angiogenesis when placed on the CALM of theembryonic chick (FIGS. 15E and 15F).

Example 12 Paclitaxel Encapsulation in Poly(E-Caprolactone)Microspheres, Inhibition of Angiogenesis on the Cam Assay byPaclitaxel-Loaded Microspheres

[0346] This example evaluates the in vitro release rate profile ofpaclitaxel from biodegradable microspheres of poly(e-caprolactone) anddemonstrates the anti-angiogenic activity of paclitaxel released fromthese microspheres when placed on the CAM.

[0347] Reagents which were utilized in these experiments include:poly(e-caprolactone) (“PCL”) (molecular weight 35,000-45,000; purchasedfrom Polysciences (Warrington, Pa.)); dichloromethane (“DCM”) fromFisher Scientific Co., Canada; polyvinyl alcohol (PVP) (molecular weight12,00-18,000, 99% hydrolysed) from Aldrich Chemical Co. (Milwaukee,Wis.), and paclitaxel from Sigma Chemical Co. (St. Louis, Mo.). Unlessotherwise stated all chemicals and reagents are used as suppliedDistilled water is used throughout.

[0348] A. Preparation of Microspheres

[0349] Microspheres are prepared essentially as described in Example 8utilizing the solvent evaporation method. Briefly, 5% w/w paclitaxelloaded microspheres are prepared by dissolving 10 mg of paclitaxel and190 mg of PCL in 2 ml of DCM, adding to 100 ml of 1% PVP aqueoussolution and stirring at 1000 rpm at 25° C. for 2 hours. The suspensionof microspheres is centrifuged at 1000 x g for 10 minutes (Beckman GPR),the supernatant removed and the microspheres washed three times withwater. The washed microspheres are air-dried overnight and stored atroom temperature. Control microspheres (paclitaxel absent) are preparedas described above. Microspheres containing 1% and 2% paclitaxel arealso prepared. Microspheres are sized using an optical microscope with astage micrometer.

[0350] B Encapsulation Efficiency

[0351] A known weight of drug-loaded microspheres (about 5 mg) isdissolved in 8 ml of acetonitrile and 2 ml distilled water is added toprecipitate the polymer The mixture is centrifuged at 1000 g for 10minutes and the amount of paclitaxel encapsulated is calculated from theabsorbance of the supernatant measured in a UV spectrophotometer(Hewlett-Packard 8452A Diode Array Spectrophotometer) at 232 nm.

[0352] C. Drug Release Studies

[0353] About 10 mg, of paclitaxel-loaded microspheres are suspended in20 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS) in screw-cappedtubes. The tubes are tumbled end-over-end at 37° C. and at given timeintervals 19.5 ml of supernatant is removed (after allowing themicrospheres to settle at the bottom). filtered through a 0.45 ummembrane filter and retained for paclitaxel analysis. An equal volume ofPBS is replaced in each tube to maintain sink conditions throughout thestudy. The filtrates are extracted with 3×1 ml DCM, the DCM extractsevaporated to dryness under a stream of nitrogen, redissolved in 1 mlacetonitrile and analyzed by HPLC using a mobile phase ofwater:methanol:acetonitrile (37:5:58) at a flow rate of 1 ml min⁻¹(Beckman Isocratic Pump), a C8 reverse phase column (Beckman), and UVdetection (Shimadzu SPD A) at 232 nm.

[0354] D. CAM Studies

[0355] Fertilized, domestic chick embryos are incubated for 4 days priorto shell-less culturing. On day 6 of incubation, 1 mg aliquots of 5%paclitaxel-loaded or control (paclitaxel-free) microspheres are placeddirectly on the CAM surface. After a 2-day exposure the vasculature isexamined using a stereomicroscope interfaced with a video camera; thevideo signals are then displayed on a computer and video printed.

[0356] E. Scanning Electron Microscopy

[0357] Microspheres are placed on sample holders, sputter-coated withgold and then placed in a Philips 501 B Scanning Electron Microscopeoperating at 15 kV.

[0358] F. Results

[0359] The size range for the microsphere samples is between 30-100 um,although there is evidence in all paclitaxel-loaded or controlmicrosphere batches of some microspheres falling outside this range. Theefficiency of loading PCL microspheres with paclitaxel is always greaterthan 95% for all drug loadings studied. Scanning electron microscopydemonstrated that the microspheres are all spherical and many showed arough or pitted surface morphology. There appeared to be no evidence ofsolid drug on the surface of the microspheres.

[0360] The time courses of paclitaxel release from 1%, 2% and 5% loadedPCL microspheres are shown in FIG. 16A. The release rate profiles arebi-phasic. There is an initial rapid release of paclitaxel or “burstphase” at all drug loadings. The burst phase occurred over 1-2 days at1% and 2% paclitaxel loading and over 3-4 days for 5% loadedmicrospheres. The initial phase of rapid release is followed by a phaseof significantly slower drug release. For microspheres containing 1% or2% paclitaxel there is no further drug release after 21 days. At 5%paclitaxel loading, the microspheres had released about 20% of the totaldrug content after 21 days.

[0361]FIG. 16B shows CAMs treated with control PCL microspheres, andFIG. 16C shows treatment with 5% paclitaxel loaded microspheres. The CAMwith the control microspheres shows a normal capillary networkarchitecture. The CAM treated with paclitaxel-PCL microspheres showsmarked vascular regression and zones which are devoid of a capillarynetwork.

[0362] G. Discussion

[0363] The solvent evaporation method of manufacturing paclitaxel-loadedmicrospheres produced very high paclitaxel encapsulation efficiencies ofbetween 95-100%. This is due to the poor water solubility of paclitaxeland its hydrophobic nature favouring partitioning in the organic solventphase containing the polymer.

[0364] The biphasic release profile for paclitaxel is typical of therelease pattern for many drugs from biodegradable polymer matrices.Poly(e-caprolactone) is an aliphatic polyester which can be degraded byhydrolysis under physiological conditions and it is non-toxic and tissuecompatible. The degradation of PCL is significantly slower than that ofthe extensively investigated polymers and copolymers of lactic andglycolic acids and is therefore suitable for the design of long-termdrug delivery systems. The initial rapid or burst phase of paclitaxelrelease is thought to be due to diffusional release of the drug from thesuperficial region of the microspheres (close to the microspheresurface). Release of paclitaxel in the second (slower) phase of therelease profiles is not likely due to degradation or erosion of PCLbecause studies have shown that under in vitro conditions in water thereis no significant weight loss or surface erosion of PCL over a 7.5-weekperiod. The slower phase of paclitaxel release is probably due todissolution of the drug within fluid-filled pores in the polymer matrixand diffusion through the pores The greater release rate at higherpaclitaxel loading is probably a result of a more extensive pore networkwithin the polymer matrix.

[0365] Paclitaxel microspheres with 5% loading have been shown torelease sufficient drug to produce extensive inhibition of angiogenesiswhen placed on the CAM. The inhibition of blood vessel growth resultedin an avascular zone as shown in FIG. 16C.

Example 13 Paclitaxel-Loaded Polymeric Films Composed of Ethylene VinylAcetate and a Surfactant

[0366] Two types of films are investigated within this example: pure EVAfilms loaded with paclitaxel and EVA/surfactant blend films loaded withpaclitaxel

[0367] The surfactants being examined are two hydrophobic surfactants(Span 80 and Pluronic L101) and one hydrophilic surfactant (PluronicF127). The pluronic surfactants are themselves polymers, which is anattractive property since they can be blended with EVA to optimizevarious drug delivery properties. Span 80 is a smaller molecule which isin some manner dispersed in the polymer matrix, and does not form ablend.

[0368] Surfactants is useful in modulating the release rates ofpaclitaxel from films and optimizing certain physical parameters of thefilms. One aspect of the surfactant blend films which indicates thatdrug release rates can be controlled is the ability to vary the rate andextent to which the compound will swell in water. Diffusion of waterinto a polymer-drug matrix is critical to the release of drug from thecarrier FIGS. 17C and 17D show the degree of swelling of the films asthe level of surfactant in the blend is altered. Pure EVA films do notswell to any significant extent in over 2 months However, by increasingthe level of surfactant added to the EVA it is possible to increase thedegree of swelling of the compound, and by increasing hydrophilicityswelling can also be increased.

[0369] Results of experiments with these films are shown below in FIGS.17A-E. Briefly, FIG. 17A shows paclitaxel release (in mg) over time frompure EVA films. FIG. 173B shows the percentage of drug remaining for thesame films. As can be seen from these two figures, as paclitaxel loadingincreases (i.e., percentage of paclitaxel by weight is increased), drugrelease rates increase, showing the expected concentration dependence.As paclitaxel loading is increased, the percent paclitaxel remaining inthe film also increases, indicating that higher loading may be moreattractive for long-term release formulations.

[0370] Physical strength and elasticity of the films is assessed in FIG.17E. Briefly, FIG. 17E shows stress/strain curves for pure EVA andEVA-Surfactant blend films. This crude measurement of stressdemonstrates that the elasticity of films is increased with the additionof Pluronic F127, and that the tensile strength (stress on breaking) isincreased in a concentration dependent manner with the addition ofPluronic F127. Elasticity and strength are important considerations indesigning a film which can be manipulated for particular clinicalapplications without causing permanent deformation of the compound.

[0371] The above data demonstrates the ability of certain surfactantadditives to control drug release rates and to alter the physicalcharacteristics of the vehicle.

Example 14 Incorporating Methoxypolyethylene Glycol 350 (MePEG) IntoPoly(E-Caprolactone) to Develop a formation for the Controlled Deliveryof Paclitaxel from a Paste

[0372] Reagents and equipment which were utilized within theseexperiments include methoxypolyethylene glycol 350 (“MePEG”—UnionCarbide, Danbury, Conn.). MePEG is liquid at room temperature, and has afreezing point of 10° to −5° C.

[0373] A. Preparation of a MePEG/PCL Paclitaxel-Containing Paste

[0374] MePEG/PCL paste is prepared by first dissolving a quantity ofpaclitaxel into MePEG, and then incorporating this into melted PCL. Oneadvantage with this method is that no DCM is required.

[0375] B. Analysis of Melting Point

[0376] The melting point of PCL/MePEG polymer blends may be determinedby differential scanning calorimetry from 30° C. to 70° C. at a heatingrate of 2.5° C. per minute. Results of this experiment are shown inFIGS. 18A and 18B. Briefly, as shown in FIG. 18A the melting point ofthe polymer blend (as determined by thermal analysis) is decreased byMePEG in a concentration dependent manner. The melting point of thepolymer blends as a function of MePEG concentration is shown in FIG.18A. This lower melting point also translates into an increased time forthe polymer blends to solidify from melt as shown in FIG. 18B. A 30:70blend of MePEG:PCL takes more than twice as long to solidify from thefluid melt than does PCL alone.

[0377] C. Measurement of Brittleness

[0378] Incorporation of MePEG into PCL appears to produce a less brittlesolid, as compared to PCL alone. As a “rough” way of quantitating this,a weighted needle is dropped from an equal height into polymer blendscontaining from 0% to 30% MePEG in PCL, and the distance that the needlepenetrates into the solid is then measured. The resulting graph is shownas FIG. 18C. Points are given as the average of four measurements +/−1S.D.

[0379] For purposes of comparison, a sample of paraffin wax is alsotested and the needle penetrated into this a distance of 7.25 mm+/−0.3mm.

[0380] D. Measurement of Paclitaxel Release

[0381] Pellets of polymer (PCL containing 0%, 5%, 10% or 20% MePEG) areincubated in phosphate buffered saline (PBS, pH 7.4) at 37° C., and %change in polymer weight is measured over time. As can be seen in FIG.18D, the amount of weight lost increases with the concentration of MePEGoriginally present in the blend. It is likely that this weight loss isdue to the release of MePEG from the polymer matrix into the incubatingfluid. This would indicate that paclitaxel will readily be released froma MePEG/PCL blend since paclitaxel is first dissolved in MePEG beforeincorporation into PCL

[0382] E. Effect of Varying Quantities of MePEG on Paclitaxel Release

[0383] Thermopastes are made up containing between 0.8% and 20% MePEG inPCL. These are loaded with 1% paclitaxel. The release of paclitaxel overtime from 10 mg pellets in PBS buffer at 37° C. is monitored using HPLC.As is shown in FIG. 18E, the amount of MePEG in the formulation does notaffect the amount of paclitaxel that is released.

[0384] F. Effect of Varying Quantities of Paclitaxel on the Total Amountof Paclitaxel Released From a 20% MePEG/PCL Blend

[0385] Thermopastes are made up containing 20% MePEG in PCL and loadedwith between 0.2% and 10% paclitaxel. The release of paclitaxel overtime is measured as described above. As shown in FIG. 18F, the amount ofpaclitaxel released over time increases with increased paclitaxelloading. When plotted as the percent total paclitaxel released, however,the order is reversed (FIG. 18G). This gives information about theresidual paclitaxel remaining in the paste and allows for a projectionof the period of time over which paclitaxel may be released from the 20%MePEG Thermopaste.

[0386] G. Strength Analysis of Various MePEG/PCL Blends

[0387] A CT-40 mechanical strength tester is used to measure thestrength of solid polymer “tablets” of diameter 0.88 cm and an averagethickness of 0.560 cm. The polymer tablets are blends of MePEG atconcentrations of 0%, 5%,10% or 20% in PCL.

[0388] Results of this test are shown in FIG. 18H, where both thetensile strength and the time to failure are plotted as a function of%MePEG in the blend. Single variable ANOVA indicated that the tabletthicknesses within each group are not different. As can be seen fromFIG. 18H, the addition of MePEG into PCL decreased the hardness of theresulting solid.

Example 15 Effect of Paclitaxel-Loaded Thermopaste on Angiogenesis InVitro

[0389] Fertilized, domestic chick embryos were incubated for 4 daysprior to shell-less culturing as described in Example 2. The eggcontents are removed from the shell and emptied into roundbottomsterilized glass bowls and covered with petri dish covers.

[0390] Paclitaxel is incorporated into thermopaste at concentrations of0.05%, 0.1%, 0.25%. 0.5%, 1.0%, 5%, 10%, and 20% (w/v) essentially asdescribed above (see Example 10), and used in the following experimentson the CAM. Dried thermopaste disks weighing 3 mg were made by heatingthe paste to 60° C., forming drop size aliquots, and then allowing it tocool.

[0391] In addition, unloaded thermopaste and thermopaste containing 20%paclitaxel were also heated to 60° C. and placed directly on the growingedge of each CALM at day 6 of incubation: two animals each were treatedin this manner. There was no observable difference in the resultsobtained using the different methods of administration indicating thatthe temperature of the paste at the time of application was not a factorin the outcome.

[0392] Each concentration of paclitaxel-loaded thermopaste (0.05%, 0.1%,0.25%, 0.5%, 1.0%, 5%, 10%, and 20%) was tested on 4 to 9 embryos at day6 of development (see Table III). After a 2 day exposure (day 8 ofincubation) the vasculature was examined with the aid of astereomicroscope. Liposyn II, a white opaque solution, was injected intothe CAM which increases the visibility of the vascular details.

[0393] The 20% paclitaxel-loaded thermopaste induced an extensive areaof avascularity (see FIG. 19B) in all 6 of the CAMs receiving thistreatment. The highest degree of inhibition was defined as a region ofavascularitv covering an area of 6 mm in diameter. All of the CAMstreated with 20% paclitaxel-loaded thermopaste displayed this degree ofangiogenesis inhibition.

[0394] In the animals treated with 5% paclitaxel-loaded paste, 4 animalsdemonstrated maximum inhibition of angiogenesis. Of the animals treatedwith 10% paclitaxel-loaded thermopaste, only 5 illustrated maximalinhibition.

[0395] The results of this study also show that paclitaxel-loadedthermopaste, as low as 0.25%, can release a significant amount of drugto induce angiogenesis inhibition on the CAM. (Table IV, FIG. 19C and19D).

[0396] By comparison, the control (unloaded) thermopaste did not inhibitangiogenesis on the CAM (see FIG. 19A); this higher magnification view(note that the edge of the paste is seen at the top of the image)demonstrates that the vessels adjacent to the paste are unaffected bythe thermopaste. This suggests that the avascular effect observed is dueto the sustained release of paclitaxel and is not due to the polymeritself or due to a secondary pressure effect of the paste on thedeveloping vasculature.

[0397] This study also demonstrates that thermopaste releases sufficientquantities of angiogenesis inhibitor (in this case paclitaxel) toinhibit the normal development of the CABS vasculature. TABLE IVAngiogenic Inhibition of Paclitaxel-Loaded Thermopaste Paclitaxel-loadedThermopaste (%) Embryos Evaluated (positive/n) 0.05 0/9 0.1 1/8 0.25 4/40.5 4/4 1 8/8 5 4/4 10 5/5 20 6/6 0 (control)  0/30

Example 16 Effect of Paclitaxel-Loaded Thermopaste on Tumor Growth andTumor Angiogenesis In Vivo

[0398] Fertilized domestic chick embryos are incubated for 3 days priorto having their shells removed. The egg contents are emptied by removingthe shell located around the airspace, severing the interior shellmembrane, perforating the opposite end of the shell and allowing the eggcontents to gently slide out from the blunted end. The contents areemptied into round-bottom sterilized glass bowls, covered with petridish covers and incubated at 90% relative humidity and 3% carbon dioxide(see Example 2).

[0399] MDAY-D2 cells (a murine lymphoid tumor) is injected into mice andallowed to grow into tumors weighing 0.5-1.0 g. The mice are sacrificed,the tumor sites wiped with alcohol, excised, placed in sterile tissueculture media, and diced into 1 mm pieces under a laminar flow hood.Prior to placing the dissected tumors onto the 9-day old chick embryos,CAM surfaces are gently scraped with a 30 gauge needle to insure tumorimplantation. The tumors are then placed on the CAMs after 8 days ofincubation (4 days after deshelling), and allowed to grow on the CAM forfour days to establish a vascular supply Four embryos are preparedutilizing this method, each embryo receiving 3 tumors. For theseembryos, one tumor receives 20% paclitaxel-loaded thermopaste, thesecond tumor unloaded thermopaste, and the third tumor no treatment. Thetreatments are continued for two days before the results were recorded.

[0400] The explanted MDAY-D2 tumors secrete angiogenic factors whichinduce the ingrowth of capillaries (derived from the CAM) into the tumormass and allow it to continue to grow in size. Since all the vessels ofthe tumor are derived from the CAM, while all the tumor cells arederived from the explant, it is possible to assess the effect oftherapeutic interventions on these two processes independently. Thisassay has been used to determine the effectiveness of paclitaxel-loadedthermopaste on: (a) inhibiting the vascularization of the tumor and (b)inhibiting the growth of the tumor cells themselves.

[0401] Direct in vivo stereomicroscopic evaluation and histologicalexamination of fixed tissues from this study demonstrated the following.In the tumors treated with 20% paclitaxel-loaded thermopaste, there wasa reduction in the number of the blood vessels which supplied the tumor(see FIGS. 20C and 20D), a reduction in the number of blood vesselswithin the tumor, and a reduction in the number of blood vessels in theperiphery of the tumor (the area which is typically the most highlyvascularized in a solid tumor) when compared to control tumors. Thetumors began to decrease in size and mass during the two days the studywas conducted. Additionally, numerous endothelial cells were seen to bearrested in cell division indicating that endothelial cell proliferationhad been affected. Tumor cells were also frequently seen arrested inmitosis. All 4 embryos showed a consistent pattern with the 20%paclitaxel-loaded thermopaste suppressing tumor vascularity while theunloaded thermopaste had no effect.

[0402] By comparison, in CAMs treated with unloaded thermopaste, thetumors were well vascularized with an increase in the number and densityof vessels when compared to that of the normal surrounding tissue, anddramatically more vessels than were observed in the tumors treated withpaclitaxel-loaded paste. The newly formed vessels entered the tumor fromall angles appearing like spokes attached to the center of a wheel (seeFIGS. 20A and 20B). The control tumors continued to increase in size andmass during the course of the study. Histologically, numerous dilatedthin-walled capillaries were seen in the periphery of the tumor and fewendothelial were seen to be in cell division. The tumor tissue was wellvascularized and viable throughout.

[0403] As an example, in two similarly-sized (initially, at the time ofexplantation) tumors placed on the same CAM the following data wasobtained. For the tumor treated with 20% paclitaxel-loaded thermopastethe tumor measured 330 mm×597 mm; the immediate periphery of the tumorhas 14 blood vessels, while the tumor mass has only 3-4 smallcapillaries. For the tumor treated with unloaded thermopaste the tumorsize was 623 mm×678 mm; the immediate periphery of the tumor has 54blood vessels, while the tumor mass has 12-14 small blood vessels. Inaddition, the surrounding CAM itself contained many more blood vesselsas compared to the area surrounding the paclitaxel-treated tumor.

[0404] This study demonstrates that thermopaste releases sufficientquantities of angiogenesis inhibitor (in this case paclitaxel) toinhibit the pathological angiogenesis which accompanies tumor growth anddevelopment. Under these conditions angiogenesis is maximally stimulatedby the tumor cells which produce angiogenic factors capable of inducingthe ingrowth of capillaries from the surrounding tissue into the tumormass. The 20% paclitaxel-loaded thermopaste is capable of blocking thisprocess and limiting the ability of the tumor tissue to maintain anadequate blood supply. This results in a decrease in the tumor mass boththrough a cytotoxic effect of the drug on the tumor cells themselves andby depriving the tissue of the nutrients required for growth andexpansion.

Example 17 Effect of Angiogenesis Inhibitor-Loaded Thermopaste on TumorGrowth In Vivo in a Murine Tumor Model

[0405] The murine MDAY-D2 tumor model may be used to examine the effectof local slow release of the chemotherapeutic and anti-angiogeniccompounds such as paclitaxel on tumor growth, tumor metastasis, andanimal survival. The MDAY-D2 tumor cell line is grown in a cellsuspension consisting of 5% Fetal Calf Serum in alpha mem media. Thecells are incubated at 37° C. in a humidified atmosphere supplementedwith 5% carbon dioxide, and are diluted by a factor of 15 every 3 daysuntil a sufficient number of cells are obtained. Following theincubation period the cells are examined by light microscopy forviability and then are centrifuged at 1500 rpm for 5 minutes. PBS isadded to the cells to achieve a dilution of 1,000,000 cells per ml.

[0406] Ten week old DBA/2j female mice are acclimatized for 3-4 daysafter arrival. Each mouse is then injected subcutaneously in theposteriolateral flank with 100,000 MDAY-D2 cells in 100 ml of PBSPrevious studies have shown that this procedure produces a visible tumorat the injection site in 3-4 days, reach a size of 1.0-1.7 g by 14 days,and produces visible metastases in the liver 19-25 days post-injection.Depending upon the objective of the study a therapeutic intervention canbe instituted at any point in the progression of the disease.

[0407] Using the above animal model, 20 mice are injected with 140,000MDAY-D2 cells s.c. and the tumors allowed to grow. On day 5 the mice aredivided into groups of 5. The tumor site was surgically opened underanesthesia, the local region treated with the drug-loaded thermopaste orcontrol thermopaste without disturbing the existing tumor tissue, andthe wound was closed. The groups of 5 received either no treatment(wound merely closed), polymer (PCL) alone, 10% paclitaxel-loadedthermopaste, or 20% paclitaxel-loaded thermopaste (only 4 animalsinjected) implanted adjacent to the tumor site. On day 16, the mice weresacrificed, the tumors were dissected and examined (grossly andhistologically) for tumor growth, tumor metastasis, local and systemictoxicity resulting from the treatment, effect on wound healing, effecton tumor vascularity, and condition of the paste remaining at theincision site.

[0408] The weights of the tumors for each animal is shown in the tablebelow: TABLE V Tumor Weights (gm) Control Control 10% Paclitaxel 20%Paclitaxel Animal No. (empty) (PCL) Thermopaste Thermopaste 1 1.3871.137 0.487 0.114 2 0.589 0.763 0.589 0.192 3 0.461 0525 0.447 0.071 40.606 0.282 0.274 0.042 5 0353 0.277 0.362 Mean 0.6808 0.6040 0.43130.1048 Std. 0.4078 0.3761 0.1202 0.0653 Deviation P Value 0.7647 0.3580.036

[0409] Thermopaste loaded with 20% paclitaxel reduced tumor growth byover 85% (average weight 0.105) as compared to control animals (averageweight 0.681). Animals treated with thermopaste alone or thermopastecontaining 10% paclitaxel had only modest effects on tumor growth; tumorweights were reduced by only 10% and 35% respectively (FIG. 21A).Therefore, thermopaste containing 20% paclitaxel was more effective inreducing tumor growth than thermopaste containing 10% paclitaxel (seeFIG. 21C; see also FIG. 21B).

[0410] Thermopaste was detected in some of the animals at the site ofadministration. Polymer varying in weight between 0.026 g to 0.078 g wasdetected in 8 of 15 mice. Every animal in the group containing 20%paclitaxel-loaded thermopaste contained some residual polymer suggestingthat it was less susceptible to dissolution. Histologically, the tumorstreated with paclitaxel-loaded thermopaste contained lower cellularityand more tissue necrosis than control tumors. The vasculature wasreduced and endothelial cells were frequently seen to be arrested incell division. The paclitaxel-loaded thermopaste did not appear toaffect the integrity or cellularity of the skin or tissues surroundingthe tumor. Grossly, wound healing was unaffected.

Example 18 The Use of Angiogenesis-Inhibitor Loaded Surgical Films inthe Prevention of Iatrogenic Metastatic Seeding of Tumor Cells DuringCancer Resection Surgery

[0411] As discussed above, sterile, pliable, stretchable drug-polymercompounds (e.g., films) may be utilized in accordance with the methodsdescribed herein in order to isolate normal surrounding tissues frommalignant tissue during resection of cancer. Such material preventsiatrogenic spread of the disease to adjacent organs through inadvertentcontamination by cancer cells. Such polymers may be particularly usefulif placed around the liver and/or other abdominal contents during bowelcancer resection surgery in order to prevent intraperitoneal spread ofthe disease to the liver.

[0412] A. Materials and Methods

[0413] Preparation of Surgical Film. Surgical films are prepared asdescribed in Example 10. Thin films measuring approximately 1 cm×1 cmare prepared containing either polymer alone (PCL) or PCL loaded with50% paclitaxel.

[0414] Rat Hepatic Tumor Model. In an initial study Wistar rats weighingapproximately 300 g underwent general anesthesia and a 3-5 cm abdominalincision is made along the midline. In the largest hepatic lobe, a 1 cmincision is made in the hepatic parenchyma and part of the liver edge isresected. A concentration of 1 million live 9L Glioma tumor cells(eluted from tissue culture immediately prior to the procedure)suspended in 100 ml of phosphate buffered saline are deposited onto thecut liver edge with a 30 gauge needle. The surgical is then placed overthe cut liver edge containing the tumor cells and affixed in place withGelfoam. Two animals received PCL films containing 5% paclitaxel and twoanimals received films containing PCL alone. The abdominal wall isclosed with 3.0 Dexon and skin clips. The general anesthetic isterminated and the animal is allowed to recover. Ten days later theanimals are sacrificed and the livers examined histologically.

[0415] B. Results

[0416] Local tumour growth is seen in the 2 livers treated with polymeralone. Both livers treated with polymer plus paclitaxel are completelyfree of tumour when examined histologically. Also of importance, theliver capsule had regenerated and grown completely over the polymericfilm and the cut surface of the liver indicating that there is nosignificant effect on wound healing. There is no evidence of localhepatic toxicity surrounding any (drug-loaded or drug-free) of thesurgical films.

[0417] C. Discussion

[0418] This study indicates that surgical films placed around normaltissues and incision sites during surgery may be capable of decreasingthe incidence of accidental implantation of tumor cells into normalsurrounding tissue during resection of malignant tumors.

Example 19 Intra-Articular Injection of Angiogenesis-Inhibitor-LoadedBiodegradable Microspheres in the Treatment of Arthritis

[0419] Articular damage in arthritis is due to a combination ofinflammation (including WBCs and WBC products) and pannus tissuedevelopment (a tissue composed on neovascular tissue, connective tissue,and inflammatory cells). Paclitaxel has been chosen for the initialstudies because it is a potent inhibitor of neovascularization. In thismanner, paclitaxel in high local concentrations will prove to be adisease modifying agent in arthritis.

[0420] In order to determine whether microspheres have a deleteriouseffect on joints, the following experiments are conducted. Briefly,plain PCL and paclitaxel-loaded microspheres are prepared as describedpreviously in Example 8. Three rabbits are injected intra-articularlywith 0.5-5.0 μm, 10-30 μm, or 30-80 μM microspheres in a total volume of0.2 mLs (containing 0.5 mg of microspheres). The joints are assessedvisually (clinically) on a daily basis. After two weeks the animals aresacrificed and the joints examined histologically for evidence ofinflammation and depletion of proteoglycans.

[0421] The rabbit inflammatory arthritis and osteoarthritis models maybe utilized in order to evaluate the use of microspheres in reducingsynovitis and cartilage degradation. Briefly, degenerative arthritis isinduced by a partial tear of the cruciate ligament and meniscus of theknee. After 4 to 6 weeks, the rabbits develop erosions in the cartilagesimilar to that observed in human osteoarthritis. Inflammatory arthritisis induced by immunizing rabbits with bovine serum albumen (BSA) inComplete Freund's Adjuvant (CFA). After 3 weeks, rabbits containing ahigh titer of anti-BSA antibody receive an intra-articular injection ofBSA (5 mg). Joint swelling and pronounced synovitis is apparent by sevendays, a proteoglycan depletion is observed by 7 to 14 days, andcartilage erosions are observed by 4 to 6 weeks.

[0422] Inflammatory arthritis is induced as described above. After 4days, the joints are injected with microspheres containing 5% paclitaxelor vehicle. One group of animals is sacrificed on day 14 and another onday 28. The joints are examined histologically for inflammation andcartilage degradation. The experiment is designed to determine ifpaclitaxel microspheres can affect joint inflammation and cartilagematrix degradation.

[0423] Angiogenesis-inhibitor microspheres may be further examined in anosteoarthritis model. Briefly, degenerative arthritis is induced inrabbits as described above, and the joints receive an intra-articularinjection of microspheres (5% paclitaxel or vehicle only) on day 4. Theanimals are sacrificed on day 21 and day 42 and the joints examinedhistologically for evidence of cartilage degradation.

[0424] Results

[0425] Unloaded PCL microspheres of differing sizes (0.5-5.0 μm, 10-30μm, or 30-80 μm) are injected intra-articularly into the rabbit kneejoint. Results of these experiments are shown in FIGS. 22A to 22DBriefly, FIG. 22A is a photograph of synovium from PBS injected joints.FIG. 22B is a photograph of joints injected with microspheres. FIG. 22Cis a photograph of cartilage from joints injected with PBS, and FIG. 22Dis a photograph of cartilage from joints injected with microspheres.

[0426] As can be seen from these photographs, histologically, there isno difference between joints receiving a microsphere injection and thosewhich did not. Clinically, there was no evidence of joint inflammationduring the 14 days the experiment was conducted. Grossly, there is noevidence of joint inflammation or cartilage damage in joints wheremicrospheres are injected, as compared to untreated normal joints.

[0427] Conclusions

[0428] Microspheres can be injected intra-articularly without causingany discernible changes to the joint surface. This indicates that thismethod may be an effective means of delivering a targeted,sustained-release of disease-modifying agents to diseased joints, whileminimizing the toxicity which could be associated with the systemicadministration of such biologically active compounds.

[0429] As discussed above, microspheres can be formulated into specificsizes with defined drug release kinetics. It has also been demonstratedthat paclitaxel is a potent inhibitor of angiogenesis and that it isreleased from microspheres in quantities sufficient to blockneovascularization on the CAM assay. Therefore,angiogenesis-inhibitor-loaded (e.g., paclitaxel-loaded) microspheres maybe administered intra-articularly in order to block theneovascularization that occurs in diseases such as rheumatoid arthritis.In this manner the drug-loaded microspheres can act as a“chondroprotective” agent which protects the cartilage from irreversibledestruction from invading neovascular pannus tissue.

Example 20 The Anti-Angiogenic Effects of Paclitaxel in an OphthalmicSuspension

[0430] In order to test whether paclitaxel would inhibit thepathogenesis of corneal neovascularization, an ophthalmic suspension of0.3% paclitaxel and a 10% paclitaxel microsphere suspension was firstprepared and tested on the CAM in order to determine whether sufficientquantities of paclitaxel could be released to inhibit angiogenesis.

[0431] Briefly, fertilized, chick eggs were incubated for 4 days priorto shell less culturing as described previously in Example 2. The eggcontents are removed from the shell and emptied into round-bottomsterilized glass bowls and covered with petri dish covers.

[0432] On day 6 of development, the ophthalmic drops were tested on theCAM. To deliver the ophthalmic suspensions, a 0.5 mL syringe was slicedinto rings measuring 1 mm thick. These rings, which formed wells whenplaced onto the CAM were used to localize a 1.5 μL aliquot of ophthalmicsuspension to the CAM's blood vessels. The paclitaxel (0.3%) suspensionwas tested on 11 embryos, whereas the 10% paclitaxel-loaded microspheresuspension was tested on 4 other embryos. The control (unloaded)ophthalmic suspension was tested on the remaining 5 CAMs. After a 48hour period, Liposyn II, a white opaque solution, was injected into theCAM which increased the visibility of the vascular details when observedwith a stereomicroscope.

[0433] Within 48 hours, the 0.3% paclitaxel suspension inhibitedangiogenesis on 11/11 CAMs tested and the 10% paclitaxel-loadedmicrosphere suspension inhibited angiogenesis on 4/4 of the embryostested. This was evident by the presence of avascular zones measuring 6mm in diameter in the vicinity of the treated area (FIG. 23A); in manycases the avascular zone exceeded the size of the application ring. Thisavascular zone was defined as a region containing disrupted blood vesselfragments and discontinuous blood flow. The functional vessels adjacentto the avascular zone were modified in such a way to redirect the bloodflow away from the drug source; these vessels possessed an angulararchitecture which was not evident in the control (unloaded) thermopastetreated CAMs.

[0434] In comparison, the control (unloaded) ophthalmic vehicle did notinhibit angiogenesis on any of the 5 CAMs tested; this was evident bythe functional vessels visible within the center of the application ring(FIG. 23B). In some cases, there was some reduction in the amount ofmicrovessels located in the control treated CAMs although this was onlydue to the aqueous suspension vehicle in which paclitaxel wasadministered.

[0435] In summary, paclitaxel was sufficiently released from theophthalmic drop suspension to inhibit angiogenesis on the CAM.Therefore, since paclitaxel can be released from this vehicle system, itmay likewise be utilized in the treatment of neovascular disease of theeye, such as corneal neovascularization.

Example 21 The Anti-Angiogenic Effects of Paclitaxel-Loaded StentCoating

[0436] A. Testing of Paclitaxel-loaded Stents on a CAM

[0437] As noted above, stents are currently used for the prevention ofluminal closure induced by a disease process, such as biliary tumoringrowth. Although stents prevent tumor ingrowth temporarily, tumoringrowth eventually recurs. In this study, strecker stents were coatedwith an EVA polymer containing paclitaxel at concentrations of 33%, 10%,and 2.5% and were tested for their ability to inhibit angiogenesis onthe CAM.

[0438] Briefly, paclitaxel-coated stent tynes (3 mm in size) were placedonto the growing vessels of the CAM at day 6 of development. Of theseCAMs, 3 received 33% paclitaxel-loaded stent coating, 5 CAMs received2.5% paclitaxel, and 1 CAM received 10% paclitaxel-loaded stent coating.In addition, control stents, coated with unloaded EVA, were tested on atotal of 6 CAMs. After 48 hours, Liposyn II was injected within the CAMto increase the vascular details during observations.

[0439] All 3 different paclitaxel concentrations of the stent coatinginhibited angiogenesis on the CAM within the 48 hour period. The CAMswere maximally inhibited which was characterized by the induction ofavascular zones measuring 6 mm in diameter (FIG. 24A).

[0440] B. Testing of Control Stents in Tumors

[0441] Similar to above, control stents were prepared as described aboveand placed into established gliosarcoma tumors of the rat liver. After a7 day period, these rats were sedated and perfused with a 2.0%glutaraldehyde in sodium cacodylate solution. The livers were excisedand the stents were dissected away from the surrounding tissue. Imagesof the cross anatomy revealed that the nylon stents had becomeincorporated into the tumor and tumor ingrowth had been establishedwithin the lumen of the stent (FIG. 25 and 26). FIG. 27 shows thatmetastasis had occurred within the lung.

[0442] Unlike the paclitaxel-loaded stent coating, the control coatingdid not inhibit angiogenesis and maintained its normal architecture inall of the 6 CAMs which were tested (FIG. 24B).

[0443] In summary, since paclitaxel coated stents have the capability ofreleasing sufficient drug to inhibit angiogenesis on the CAM, paclitaxelcoated stents may likewise be utilized for a variety of applications inorder to prevent tumor ingrowth within the binary lumen.

Example 22 Effect of Paclitaxel on Neutrophil Activity

[0444] The example describes the effect of paclitaxel on the response ofneutrophils stimulated with opsonized CPPD crystals or opsonizedzymosan. As shown by experiments set forth below, paclitaxel is a stronginhibitor of particulate inducted neutrophil activation as measured bychemiluminescence, superoxide anion production and degranulation inresponse to plasma opsonized microcrystals or zymosan.

[0445] A. Materials and Methods

[0446] Hanks buffered salt solution (HBSS) pH 7.4 was used throughoutthis study. All chemicals were purchased from Sigma Chemical Co (St.Louis,) unless otherwise stated. All experiments were performed at 37° Cunless otherwise stated.

[0447] 1. Preparation and Characterization of Crystals

[0448] CPPD (triclinic) crystals were prepared. The size distribution ofthe crystals was approximately 33% less than 10 μm, 58% between 10 and20 μm and 9% greater than 20 μm. Crystals prepared under the aboveconditions are pyrogen free and crystals produced under sterile, pyrogenfree conditions produced the same magnitude of neutrophil response ascrystals prepared under normal, non-sterile laboratory conditions.

[0449] 2. Opsonization of Crystals and Zymosan

[0450] All experiments that studied neutrophil responses to crystals orzymosan in the presence of paclitaxel were performed using plasmaopsonized CPPD or zymosan. Opsonization of crystals or zymosan was donewith 50% heparinized plasma at a concentration of 75 mg of CPPD or 12 mgof zymosan per ml of 50% plasma. Crystals or zymosan were incubated withplasma for 30 min. at 37° C. and then washed in excess HBSS.

[0451] 3. Neutrophil Preparation

[0452] Neutrophils were prepared from freshly collected human citratedwhole blood. Briefly, 400 ml of blood were mixed with 80 ml of 4%dextran T500 (Phamacia LKB, Biotechnology AB Uppsala, Sweden) in HBSSand allowed to settle for 1 h. Plasma was collected continuously and 5ml applied to 5 ml of Ficoll Paque (Pharmacia) in 15 ml polypropylenetubes (Coming, N.Y.). Following centrifugation at 500×g for 30 min, theneutrophil pellets were washed free of erythrocytes by 20 s of hypotonicshock. Neutrophils were resuspended in HBSS kept on ice and used forexperiments within 3 h. Neutrophil viability and purity was alwaysgreater than 90%.

[0453] 4. Incubation of Neutrophils with Paclitaxel

[0454] A stock solution of paclitaxel at 12 mM in DMSO was freshlyprepared before each experiment. This stock solution was diluted in DMSOto give solutions of paclitaxel in the 1 to 10 mM concentration range.Equal volumes of these diluted paclitaxel solutions was added toneutrophils at 5,000,000 cells per ml under mild vortexing to achieveconcentrations of 0 to 50 μM with a final DMSO concentration of 0.5%.Cells were incubated for 20 minutes at 33° C. then for 10 minutes at 37°C. before addition to crystals or zymosan.

[0455] 5. Chemiluminescence Assay

[0456] All chemiluminescence studies were performed at a cellconcentration of 5,000,000 cells/ml in HBSS with CPPD (50 mg/ml). In allexperiments 0.5 ml of cells was added to 25 mg of CPPD or 0 5 mg ofzymosan in 1.5 ml capped Eppendorf tubes. 10 μl of luminol dissolved in25% DMSO in HBSS was added to a final concentration of 1 μM and thesamples were mixed to initiate neutrophil activation by the crystals orzymosan. Chemiluminescence was monitored using an LKB Luminometer (Model1250) at 37° C. with shaking immediately prior to measurements toresuspend the crystals or zymosan. Control tubes contained cells, drugand luminol (crystals absent).

[0457] 6 Superoxide Anion Generation

[0458] Superoxide anion concentrations were measured using thesuperoxide dismutase inhibitable reduction of cytochrome c assay.Briefly, 25 mg of crystals or 0.5 mg of zymosan was placed in a 1.5 mlcapped Eppendorf tube and warmed to 37° C. 0.5 ml of cells at 37° C.were added together with ferricytochrome c (final concentration 1.2mg/ml) and the cells were activated by shaking the capped tubes. Atappropriate times tubes were centrifuged at 10,000 g for 10 seconds andthe supernatant collected for assay be measuring the absorbance of 550nm. Control tubes were set up under the same conditions with theinclusion of superoxide dismutase at 600 units per ml.

[0459] 7. Neutrophil Degranulation Assay

[0460] One and a half milliliter Eppendorf tubes containing either 25 mgof CPPD or 1 mg of zymosan were preheated to 37° C. 0.5 ml of cells at37° C. were added followed by vigorous shaking to initiate thereactions. At appropriate times, tubes were centrifuged at 10,000×g for10 seconds and 0.4 ml of supernatant was stored at −20° C. for laterassay.

[0461] Lysozyme was measured by the decrease in absorbance at 450 nm ofa Micrococcus lysodeikticus suspension. Briefly, Micrococcuslysodeikticus was suspended at 0.1 mg/ml in 65 mM potassium phosphatebuffer, pH 6.2 and the absorbance at 450 nm was adjusted to 0.7 units bydilution. The crystal (or zymosan) and cell supernatant (100 μl) wasadded to 2.5 ml of the Micrococcus suspension and the decrease inabsorbance was monitored. Lysozyme standards (chicken egg white) in the0 to 2000 units/ml range were prepared and a calibration graph oflyzozyme concentration against the rate of decrease in the absorbance at450 nm was obtained.

[0462] Myeloperoxidase (MPO) activity was measured by the increase inabsorbance at 450 nm that accompanies the oxidation of dianisidine. 7.8mg of dianisidine was dissolved in 100 ml of 0.1M citrate buffer, pH 5.5at 3.2 mM by sonication. To a 1 ml cuvette, 0.89 mL of the dianisidinesolution was added, followed by 50 μl of 1% Triton×100, 10 μl of a 0.05%hydrogen peroxide in water solution and 50 ul of crystal-cellsupernatant. M?O activity was determined from the change in absorbance(450 nm) per minute, Delta A 450, using the following equation:

Dianisidine oxidation (nmol/min)=50×Delta A 450

[0463] 8. Neutrophil Viability

[0464] To determine the effect of paclitaxel on neutrophil viability therelease of the cytoplasmic marker enzyme, Lactate Dehydrogenase (LDH)was measured. Control tubes containing cells with drug (crystals absent)from degranulation experiments were also assayed for LDH.

[0465] B. Results

[0466] In all experiments statistical significance was determined usingStudents' t-test and significance was claimed at p<0.05. Where errorbars are shown they describe one standard deviation about the mean valuefor the n number given.

[0467] 1. Neutrophil Viability

[0468] Neutrophils treated with paclitaxel at 46 μM for one hour at 37°C. did not show any increased level of LDH release (always less than 5%of total) above controls indicating that paclitaxel did not cause celldeath.

[0469] 2. Chemiluminescence

[0470] Paclitaxel at 28 μM produced strong inhibition of both plasmaopsonized CPPD and plasma opsonised zymosan induced neutrophilchemiluminescence as shown in FIGS. 29A, 29B and 33A respectively. Theinhibition of the peak chemiluminescence response was 52% (+/−12%o) and45% (+/−11%) for CPPD and zymosan respectively. The inhibition bypaclitaxel at 28 μM of both plasma opsonized CPPD and plasma opsonizedzymosan induced chemiluminescence was significant at all times from 3 to16 minutes (FIGS. 29A, 29B and 33A). FIGS. 29A and 29B show theconcentration dependence of paclitaxel inhibition of plasma opsonizedCPPD induced neutrophil chemiluminescence. In all experiments controlsamples never produced chemiluminescence values of greater than 5 mV andthe addition of paclitaxel at all concentrations used in this study hadno effect on the chemiluminescence values of controls.

[0471] 3. Superoxide Generation

[0472] The time course of plasma opsonised CPPD crystal inducedsuperoxide anion production, as measured by the superoxide dismutase(SOD) inhibitable reduction of cytochrome c, is shown in FIG. 2.Treatment of the cells with paclitaxel at 28 μM produced a decrease inthe amount of superoxide generated at all times. This decrease wassignificant at all times shown in FIG. 30A. The concentration dependenceof this inhibition is shown in FIG. 30B. Stimulation of superoxide anionproduction by opsonised zymosan (FIG. 31B) showed a similar time courseto CPPD induced activation. The inhibition of zymosan induced superoxideanion production by paclitaxel at 28 μM was less dramatic than theinhibition of CPPD activation but was significant at all times shown inFIG. 31B.

[0473] 4. Neutrophil Degranulation

[0474] Neutrophil degranulation was monitored by the plasma opsonizedCPPD crustal induced release of myeloperoxidase and lysozyme or theplasma opsonized zymosan induced release of myeloperoxidase. It has beenshown that sufficient amounts of these two enzymes are released into theextracellular media when plasma coated CPPD crystals are used tostimulate neutrophils without the need for the addition of cytochalasinB to the cells. FIGS. 32 and 33 show the time course of the release ofMPO and lysozyme respectively, from neutrophils stimulated by plasmacoated CPPD. FIG. 32A shows that paclitaxel inhibits myeloperoxidaserelease from plasma opsonized CPPD activated neutrophils in the first 9minutes of the crystal-cell incubation. Paclitaxel significantlyinhibited CPPD induced myeloperoxidase release at all times as shown inFIG. 32A. FIG. 32B shows the concentration dependence of paclitaxelinhibition of CPPD induced myeloperoxidase release.

[0475] Paclitaxel at 28 μM reduced lysozyme release and this inhibitionof degranulation was significant at all times as shown in FIG. 33.

[0476] Only minor amounts of MPO and lysozyme were released whenneutrophils were stimulated with opsonized zymosan. Despite these lowlevels it was possible to monitor 50% inhibition of MPO release after 9minutes incubation in the presence of paclitaxel at 28 μM that wasstatistically significant (p<0.05) (data not shown).

[0477] C. Discussion

[0478] These experiments demonstrate that paclitaxel is a stronginhibitor of crystal induced neutrophil activation. In addition, byshowing similar levels of inhibition in neutrophil responses to anotherform of particulate activator, opsonized zymozan, it is evident that theinhibitory activity of paclitaxel is not limited to neutrophil responsesto crystals.

Example 23 Effect of Paclitaxel on Synoviocyte Proliferation

[0479] Two experiments were conducted in order to assess the effect ofdiffering concentrations of paclitaxel on tritiated thymidineincorporation (a measurement of synoviocyte DNA synthesis) andsynoviocyte proliferation in vitro.

[0480] A. Materials and Methods

[0481] 1. ³H-Thymidine Incorporation into Synoviocytes

[0482] Synoviocytes were incubated with different concentrations ofpaclitaxel (10⁻⁵ M, 10 ⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M) continuously for 6 or24 hours in vitro. At these times, 1×10⁻⁶ cpm of ³H-thymidine was addedto the cell culture and incubated for 2 hours at 37° C. The cells wereplaced through a cell harvester, washed through a filter, the filterswere cut, and the amount of radiation contained in the filter sectionsdetermined. Once the amount of thymidine incorporated into the cells wasascertained, it was used to determine the rate of cell proliferation.This experiment was repeated three times and the data collated together.

[0483] 2. Synoviocyte Proliferation

[0484] Bovine synovial fibroblasts were grown in the presence andabsence of differing concentrations (10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M and 10⁻⁸ M)of paclitaxel for 24 hours. At the end of this time period the totalnumber of viable synoviocyte cells was determined visually by dyeexclusion counting using Trypan blue staining. This experiment wasconducted 4 times and the data collated.

[0485] B. Results

[0486] 1. ³H-7Thymidine Incorporation into Synoviocytes

[0487] This study demonstrated that paclitaxel at low concentrationsinhibits the incorporation of 3H-thymidine (and by extension DNAsynthesis) in synoviocytes at concentrations as low as 10⁻⁸ M. At sixhours there was no significant difference in the between the degree ofinhibition produced by the higher versus the lower concentrations ofpaclitaxel (see FIG. 34). However, by 24 hours some of the effect waslost at lower concentrations of the drug (10⁻⁸ M), but was stillsubstantially lower than that seen in control animals.

[0488] 2. Synoviocyte Proliferation

[0489] This study demonstrated that paclitaxel was cytotoxic toproliferating synovial fibroblasts in a concentration dependent manner.Paclitaxel at concentrations as low as 10⁻⁷ M is capable of inhibitingproliferation of the synovioctes (see FIG. 35). At higher concentrationsof paclitaxel (10⁻⁶ M and 10⁻⁵ M) the drug was toxic to the synovialfibroblasts in vitro.

[0490] C. Discussion

[0491] The above study demonstrates that paclitaxel is capable ofinhibiting the proliferation of synovial fibroblasts at relatively lowconcentrations in vitro. Therefore, given the role of these cells in thedevelopment of pannus tissue and their growth during the pathogenesis ofrheumatoid arthritis, blocking synoviocyte proliferation can be expectedto favorably affect the outcome of the disease in vivo.

Example 24 Effect of Paclitaxel on Collegenase Expression

[0492] As noted above, collagenase production by a variety of tissues(synovial fibroblasts, endothelial cells, chondrocytes, and white bloodcells) plays an critical role in the development of the pathology ofarthritis. Degradation of the cartilage matrix by proteolytic enzymesrepresents an irreversible step in the development of the diseaseresulting in irreparable damage to the articular cartilage. Numerousattempts have been made to restore the balance between the enzymes whichdegrade connective tissue (matrix metalloproteinases—MMPs; collagenaseis an important member of this family) and those which inhibitdegradation (tissue inhibitors of metalloproteinases—TMPs). Evidencesuggests that the imbalance of proteolytic versus inhibitory activitywhich results in cartilage destruction is due to an excess of MMPactivity as opposed to a paucity of TIMP activity. Treatment thatdecreases the amount of MMP activity may thus favorably influence theoutcome of the disease.

[0493] C-fos is an oncogene transcription factor shown to be involvedand required for the induction of genes involved in cell proliferationand collagenase expression. In cultured chondrocytes, both interleukin-1(IL-1) and tumor necrosis factor (TNF) have been shown to stimulatec-fos expression and produce all of the signals necessary to induce theexpression of collagenase. When IL-1 is administered to chondrocytes invitro there is a transient increase in fos mRNA levels which peak 30-60minutes later, while collagenase mRNA is detected 9 hours later andcontinues to increase up to 12 hours (data not shown) after IL-1stimulation. The fos and collagenase mRNA can be detected using therespective cDNA probes and analyzed by Northern blot analysis. Thisallows the determination of agents capable of inhibiting collagenaseproduction and an approximation of the step in the collagenase syntheticpathway that is affected by the treatment.

[0494] A. Materials and Methods

[0495] 1. Effect of Paclitaxel on c-fos Expression

[0496] Chondrocytes were treated with different concentrations ofpaclitaxel (10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M) for 2 hours and then treatedwith TNFα (Sigma Chemical Co., St. Louis, Mo.) at 30 ng/ml for 1 hour.Human recombinant TNFα was dissolved in phosphate buffered saline (PBS)with 0.1% bovine serum albumin (BSA). Total RNA from bovine articularchondrocytes was isolated by the acidified guanidine isothiocyanatemethod and the levels of c-fos mRNA determined by Northern blotanalysis. Denatured RNA samples (12 μg) were analyzed by gelelectrophoresis in a denaturing 1% agarose gel, transferred to a nylonmembrane (Bio-Rad), cross-linked with an ultraviolet cross-linker(Stratagene UV stratalinker 1800), and hybridized with ³²P-labeled ratc-fos DNA. mRNA for tubulin and total RNA were used as controls. Todetermine tubulin mRNA, the blots described above were subsequentlystripped of DNA and re-probed with ³²P-labeled rat P-tubulin cDNA. Thisexperimented was conducted three times and the data collated.

[0497] 2. Effect of Paclitaxel on Collagenase Expression

[0498] Chondrocytes were treated with different concentrations ofpaclitaxel (10⁻⁶ M and 10⁻⁷ M,) for 2 hours prior to the addition ofIL-1 (20 ng/ml). The cells were then incubated for a further 16 hours.Total RNA from bovine articular chondrocytes was isolated by theacidified guanidine isothiocyanate method and the collagenase mRNAdetermined by Northern blot analysis. The RNA samples were prepared asdescribed above using ³²P-labeled rat collagenase cDNA.

[0499] B. Results

[0500] 1. Effect of Paclitaxel on c-fos Expression

[0501] This experiment demonstrates that paclitaxel does not alter c-fosexpression at any concentration (see FIG. 36). Comparable levels ofc-fos mRNA were detectable in the controls and all of the experimentalgroups regardless of the paclitaxel concentration present. Total RNA andtubulin expression was similarly unaffected.

[0502] 2. Effect of Paclitaxel on Collagenase Expression

[0503] This experiment demonstrates that paclitaxel at a concentrationof 10⁻⁶ M completely inhibited IL-1 induced collagenase expression.Collagenase mRNA was not detectable above background at thisconcentration of paclitaxel in vitro (see FIG. 37).

[0504] C. Discussion

[0505] Paclitaxel is capable of inhibiting collagenase production bychondrocytes in vitro at concentrations of 10⁻⁶M. This inhibition occursdownstream from the transcription factor activity of c-fos, but stillrepresents a secondary gene response, as collagenase mRNA production isaffected. As such, paclitaxel inhibition of collagenase production isnot strictly due to interruption of the microtubules involved in theprotein secretory pathway (which is dependent upon microtubular functionfor the movement of secretory granules), but acts at the level of thegene response to stimulation of collagenase production. Regardless ofthe mechanism of action, paclitaxel is capable of inhibiting collagenaseproduction by at least one cell type known to produce this enzyme in thearthritic disease process.

Example 25 Effect of Paclitaxel on Chondrocyte Viability

[0506] While it is important that a disease modifying agent be capableof strongly inhibiting a variety of inappropriate cellular activities(proliferation, inflammation, proteolytic enzyme production) which occurin excess during the development of RA, it must not be toxic to thenormal joint tissue. It is particularly critical that normalchondrocytes not be damaged, as this would hasten the destruction of thearticular cartilage and lead to progression of the disease. In thisexample, the effect of paclitaxel on normal chondrocyte viability invitro was examined.

[0507] Briefly, chondrocytes were incubated in the presence (₁₀ ⁻⁵ M,10⁻⁷ M, and 10⁻⁹ M) or absence (control) of paclitaxel for 72 hours. Atthe end of this time period, the total number of viable chondrocytes wasdetermined visually by dye exclusion counting using Trypan bluestaining. This experiment was conducted 4 times and the data collated.

[0508] Results of this experiment are shown in FIG. 38. Briefly, as isevident from FIG. 38, paclitaxel does not affect the viability of normalchondrocytes in vitro even at high concentrations (10⁻⁵ M) ofpaclitaxel. More specifically, even at drug concentrations sufficient toblock the pathological processes described in the preceding examples,there is no cytotoxicity to normal chondrocytes

Example 26 Ophthalmic Drops Containing Paclitaxel or PrednisoloneAcetate

[0509] Three formulations containing 0.3% paclitaxel for ophthalmic usewere prepared. The particle size distribution of paclitaxel as receivedfrom a supplier was not within acceptable limits for ophthalmic use. Inparticular, for ophthalmic drops at least 90% of the particles shouldpreferably be below 10 μm, with no particle above 20 μm. Two methodswere used to reduce the particle size. The first method involvedprecipitating paclitaxel from its solution in acetone. Briefly. 150 mgof paclitaxel was dissolved in 5 ml of acetone. This solution was addedin a gentle stream, with stirring, to 20 ml of Sterile water USP toprecipitate the drug. The suspension was homogenized with the Douncehomogenizer until about 90% of the drug was under 10 μm. The suspensionwas allowed to stand for about 1 hour. The larger particles settled andwere separated from the smaller ones by decantation. The largerparticles were again reduced until all particles were under 20 μm (seeFIG. 48). This suspension was added to the one previously decanted andthe acetone was evaporated by heating at 50° C. for 2 hours and then ina vacuum oven at 30° C. and 25 torr overnight to remove the residualacetone. Sodium chloride (0.45g) was dissolved in 5 ml Sterile waterUSP. This solution and 20 ml of 5% PVA solution were mixed with thepaclitaxel suspension, made up to 50 ml with sterile water and bottled.

[0510] Paclitaxel suspension (0.3%) was also prepared by adding 150 mgof paclitaxel to 10 ml of sterile water and comminuted using the FritschPulverizer for 15 minutes. It was not possible to produce particleslower than 60 μm with this method probably because the solid paclitaxelwas not hard and brittle (see FIG. 49). The suspension was mixed with 5ml sterile water containing 0.45 of NaCl and 20 ml of 5% PVA solutionand made up to 50 ml with sterile water.

[0511] Paclitaxel microspheres containing 10% paclitaxel in PCL werealso prepared. Briefly, paclitaxel (60 mg) and PCL (540 mg) weredissoived in 3 ml of DCM. 20 ml of 3% PVA solution was added andhomogenized with the Polytron homogenizer at point 3 setting for about 1minute. The emulsion was poured into a 30 ml beaker and stirred untilthe microspheres were formed (about 3 hours). This suspension was placedin a vacuum oven, at 30° C. and 25 torr, overnight to remove residualDCM. The small microsphere suspension (15 ml) was decanted andevaporated under vacuum to about 5 ml and assayed for paclitaxel. Thissuspension was mixed with 2 ml solution of NaCl (0.45 g) solution andmade up to 10 ml with sterile water.

[0512] Prednisolone acetate suspension containing 1% drug was preparedby homogenizing the appropriate amount of the drug (as received) in 20ml of 5% PVA, NaCl solution was added and made up to volume with sterilewater.

Example 27 Paclitaxel in an Animal Model of Corneal Neovascularization

[0513] Induction of Corneal Neovascularization

[0514] Corneal angiogenesis is induced in male New Zealand white rabbits(2.5 to 3.0 kg) essentially as described by Scroggs et al., Invest.Ophthalmol. Vis. Sci. 32:2105-2111, 1991. Briefly, rabbits areanesthetized with a subcutaneous injection of 0.15 cc of a 1:1 mixtureof ketamine (80 mg/ml) and xylazine (4 mg/ml), and the eyes cauterizedby applying the tip of a new silver-potassium nitrate applicator (75%silver nitrate: 25% potassium nitrate: Graham-Field, Hauppage, N.Y.) 3to 4 millimeters from the corneo-scleral limbus.

[0515] Immediately following chemical cauterization, one drop of thestudy solution (e.g., the study solutions may be vehicle alone,prednisolone acetate 10/0. or 0.3% paclitaxel in suspension) is appliedto the cauterized eyes. Gentamicin ophthalmic ointment is then appliedto the treated eyes. Over the next two weeks, one drop of the studysolution is applied four times daily.

[0516] In a second study, 0.5 ml aliquots of a 10% paclitaxel-loadedmicrosphere suspension and a 20% paclitaxel-loaded thermopaste isadministered via subconjuctival injection to the experimental animals.

[0517] On the eighth day and fourteenth following cauterization of thecorneas, all animals are re-anesthetized as described above, and thecorneas photographed using a Nikon biomicroscope and Kodak ASA 180tungsten film under microscope incandescent illumination. The highestmagnification that incorporates the entire cornea is used.

[0518] The photographs are randomly presented to a masked observer whogrades the corneal vessels based upon a 0 to 4 scale of vessel density,and who measures the total extent in clock hours of circumferentialcorneal neovascularization. Vessel density grade is based on twostandard photographs obtained from pilot experiments that had beenassigned grades 2 (moderate vessel density) and 4 (severe vesseldensity) respectively. Grades 1 and 3 were established be interpolation;grade 0 is applied to corneas that demonstrate a central cautery scar,but the absence of new vessel growth.

[0519] Differences in both corneal vessel density and extent, in termsof clock hours of involvement, is analyzed using non-paired Student's ttests. Tests are two-tailed, with a p value of ≦0.05 consideredsignificant. Measures are reported as mean ±standard deviation.

Example 28 Modification of Paclitaxel Release from Thermopaste Using LowMolecular Weight Poly(D,L, Lactic Acid)

[0520] As discussed above, depending on the desired therapeutic effect,either quick release or slow release polymeric carriers may be desired.For example, polycaprolactone (PCL) and mixtures of PCL withpoly(ethylene glycol) (PEG) produce compositions which releasepaclitaxel over a period of several months. In particular, the diffusionof paclitaxel in the polymers is very slow due to its large molecularsize and extreme hydrophobicity

[0521] On the other hand, low molecular weight poly(DL-lactic acid)(PDLLA) gives fast degradation, ranging from one day to a few monthsdepending on its initial molecular weight. The release of paclitaxel, inthis case, is dominated by polymer degradation. Another feature of lowmolecular weight PDLLA is its low melting temperature, (i.e., 40° C.-60°C.), which makes it suitable material for making Thermopaste Asdescribed in more detail below, several different methods can beutilized in order to control the polymer degradation rate, including,for example, by changing molecular weight of the PDLLA, and/or by mixingit with high mol wt. PCL, PDLLA or poly(lactide-co-glyocide) (PLGA).

[0522] A. Experimental Materials

[0523] D,L-lactic acid was purchased from Sigma Chemical Co., St. Louis,Mo. PCL (molecular weight 10-20,000) was obtained from Polysciences,Warrington, Pa. High molecular weight PDLLA (intrinsic viscosity 0.60dl/g) and PLGA (50:50 composition, viscosity 0.58 dl/g) were fromBirmingham Polymers.

[0524] B. Synthesis of Low Molecular weight PDLLA

[0525] Low molecular weight PDLLA was synthesized from DL-lactic acidthrough polycondensation. Briefly, DL-lactic acid was heated in a glassbeaker at 200° C. with nitrogen purge and magnetic stirring for adesired time. The viscosity increased during the polymerization, due tothe increase of molecular weight. Three batches were obtained withdifferent polymerization times, i.e., 40 min (molecular weight 800), 120min, 160 min.

[0526] C. Formulation of Paclitaxel Thermopastes

[0527] Paclitaxel was loaded, at 20%, into the following materials byhand mixing at a temperature about 60° C.

[0528] 1. low molecular weight PDLLA with polymerization time of 40 min.

[0529] 2. low molecular weight PDLLA with polymerization time of 120min.

[0530] 3. low mol. wt PDLLA with polymerization time of 160 min.

[0531] 4. a mixture of 50:50 high molecular weight PDLLA and lowmolecular weight PDLLA 40 min.

[0532] 5 a mixture of 50:50 high molecular weight PLGA and low molecularweight PDLLA 40 min.

[0533] 6 mixtures of high molecular weight PCL and low molecular weight.PDLLA 40min with PCL.PDLLA of 10:90, 20:80, 40:60, 60:40, and 20:80.Mixtures of high molecular weight PDLLA or PLGA with low molecularweight. PDLLA were obtained by dissolving the materials in acetonefollowed by drying.

[0534] D Release Study

[0535] The release of paclitaxel into PBS albumin buffer at 37° C. wasmeasured as described above with HPLC at various times.

[0536] E. Results

[0537] Low molecular weight PDLLA 40 min was a soft material with lightyellow color. The color is perhaps due to the oxidation during thepolycondensation. Low molecular weight PDLLA 120 min (yellow) and 160min (brown) were brittle solids at room temperature. They all becomemelts at 60° C. Mixtures of 50:50 high molecular weight PDLLA or PLGAwith low molecular weight PDLLA 40 min also melted about 60° C.

[0538] During the release, low molecular weight PDLLA 40 min and 120 minbroke up into fragments within one day, other materials were intact upto this writing (3 days).

[0539] The release of paclitaxel from formulations 2-5 were shown inFIG. 50. Low molecular weight PDLLA 40 min and 120 min gave the fastestrelease due to the break up of the paste. The release was perhapssolubility limited. Low molecular weight PDLLA 160 min. also gave a fastrelease yet maintained an intact pellet. For example, 10% of loadedpaclitaxel was released with one day. The 50:50 mixtures of highmolecular weight PDLLA or PLGA with low molecular weight PDLLA 40 minwere slower, i.e., 3.4% and 2.2% release within one day.

[0540] Although not specifically set forth above, a wide variety ofother polymeric carriers may be manufactured, including for example, (1)low molecular weight (500-10,000) poly(D,L-lactic acid), poly(L-lacticacid), poly(glycolic acid), poly(6-hydroxycaproic acid),poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), and theircopolymers; (2) blends of above (#1) above, (3) blends of (#1) abovewith high molecular weight poly(DL-lactic acid), poly(L-lactic acid),poly(glycolic acid), poly(6-hydroxycaproic acid), poly(5-hydroxyvalericacid), poly(4-hydroxybutyric acid), and their copolymers; and (4)copolymers of poly(ethylene glycol) and pluronics with poly(D,L-lacticacid), poly(L-lactic acid), poly(glycolic acid), poly(6-hydroxycaproicacid), poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), andtheir copolymers.

Example 29 Surfactant Coated Microspheres

[0541] A. Materials and Methods

[0542] Microspheres were manufactured from Poly (DL) lactic acid (PLA),poly methylmethacrylate (PMMA), polycaprolactone (PCL) and 50:50Ethylene vinyvl acetate (EVA):PLA essentially as described in Example 8.Size ranged from 10 to 100 um with a mean diameter 45 um.

[0543] Human blood was obtained from healthy volunteers. Neutrophils(white blood cells) were separated from the blood using dextransedimentation and Ficoll Hypaque centrifugation techniques. Neutrophilswere suspended at 5 million cells per ml in Hanks Buffered Salt Solution(“HBSS”).

[0544] Neutrophil activation levels were determined by the generation ofreactive oxygen species as determined by chemiluminescence. Inparticular, chemiluminescence was determined by using an LKB luminometerwith 1 uM luminol enhancer. Plasma precoating (or opsonization) ofmicrospheres was performed by suspending 10 mg of microspheres in 0.5 mlof plasma and tumbling at 37° C. for 30 min.

[0545] Microspheres were then washed in 1 ml of HBSS and the centrifugedmicrosphere pellet added to the neutrophil suspension at 37° C. at timet=0. Microsphere surfaces were modified using a surfactant calledPluronic F127 (BASF) by suspending 10 mg of microspheres in 0.5 ml of 2%w/w solution of F127 in HBSS for 30 min at 37° C. Microspheres were thenwashed twice in 1 ml of HBSS before adding to neutrophils or to plasmafor further precoating.

[0546] B. Results

[0547]FIG. 51 shows that the untreated microspheres givechemiluminescence values less than 50 mV These values represent lowlevels of neutrophil activation. By way of comparison, inflammatorymicrocrystals might give values close to 1000 mV, soluble chemicalactivators might give values close to 5000 mV. However, when themicrospheres are precoated with plasma, all chemiluminescence values areamplified to the 100 to 300 mV range (see FIG. 51). These levels ofneutrophil response or activation can be considered mildly inflammatory.PEA gave the biggest response and could be regarded as the mostinflammatory. PLA and PCL both become three to four times more potent inactivating neutrophils after plasma pretreatment (or opsonization) butthere is little difference between the two polymers in this regard.EVA:PLA is not likely to be used in angiogenesis formulations since themicrospheres are difficult to dry and resuspend in aqueous buffer. Thiseffect of plasma is termed opsonization and results from the adsorptionof antibodies or complement molecules onto the surface. These adsorbedspecies interact with receptors on white blood cells and cause anamplified cell activation.

[0548] FIGS. 52-55 describe the effects of plasma precoating of PCL,PMMA, PLA and EVA:PLA respectively as well as showing the effect ofpluronic F127 precoating prior to plasma precoating of microspheres.These figures all show the same effect: (1) plasma precoating amplifiesthe response; (2) Pluronic F127 precoating has no effect on its own, (3)the amplified neutrophil response caused by plasma precoating can bestrongly inhibited by pretreating the microsphere surface with 2%pluronic F127.

[0549] The nature of the adsorbed protein species from plasma was alsostudied by electrophoresis. Using this method, it was shown thatpretreating the polymeric surface with Pluronic F127 inhibited theadsorption of antibodies to the polymeric surface.

[0550] FIGS. 56-59 likewise show the effect of precoating PCL, PMMA, PLAor EVA:PLA microspheres (respectively) with either IgG (2 mg/ml) or 2%pluronic F127 then IgG (2 mg/ml). As can be seen from these figures, theamplified response caused by precoating microspheres with IgG can beinhibited by treatment with pluronic F127.

[0551] This result shows that by pretreating the polymeric surface ofall four types of microspheres with Pluronic F127, the “inflammatory”response of neutrophils to microspheres may be inhibited.

Example 30 Preparation of Low Molecular Weight Poly(D,L-Lactic Acid)

[0552] Five hundred grams of D,L-lactic acid (Sigma Chemical Co., St.Louis, Mo.) was heated in a heating mantle at 190° C. for 90 minutesunder a stream of nitrogen gas. This process produced 400 g ofpoly(D,L-lactic acid) with a molecular weight of 700-800 as determinedby end group titration and gel permeation chromatography (Fukusaki etal, Eur. Polym. J. 25(10):1019- 1026, 1989).

Example 31 Preparation of Polymeric Compositions Containing GelatinizedPaclitaxel

[0553] A. Preparation of Polymers

[0554] Two hundred milligrams of gelatin (Type B, bloom strength 225,Fisher Scientific) 200 mg of NaCl or 100 mg of gelatin and 100 mg ofNaCl were dissolved in 0.5 mL of water. Next, 200 mg of paclitaxel wasdissolved in 0.5 mL of ethanol. The dissolved gelatin, salt, or gelatinand salt were then added to the paclitaxel and triturated on a petridish incubating in a water bath at 80° C., until dry. The precipitatewas then ground in a mortar and pestle and sieved through either no. 60or no. 140 mesh (Endecott, London, England). (No. 60 mesh produceslarger granules and no. 140 mesh produces smaller granules.)Polycaprolactone was then heated to 60° C., and granules added to afinal ratio of 40:60 (w/w). The polymeric composition was placed into a1 ml syringe and extruded.

[0555] B. Analysis of Paclitaxel Release

[0556] A measured amount of the cylindrical polymeric composition isthen added into an albumin buffered solution, and, over a time course,aliquots are removed and paclitaxel extracted with DCM. The extracts arethen analyzed by HPLC. Results of these experiments are shown in FIGS.39 and 40. Briefly, FIG. 38 shows a greater percentage of paclitaxelreleased when large gelatinized particles (>200 μm) are utilized. FIG.39 shows that addition of NaCl is not preferred when higher amounts ofpaclitaxel release is desired.

Example 32 Copolymerization of Poly(D,L-Lactic Acid) Polyethylene Glycol

[0557] D,L,lactide (Aldrich Chemical Co.) was added to polyethyleneglycol (molecular weight 8,000;, Sigma Chemical Co., St. Louis, Mo.) ina tube and heated with 0.5% stannous octoate (Sigma Chemical Co.) for 4hours at 150° C. in an oven.

[0558] This process produces a copolymer of poly(D,L-lactic acid) withpolyethylene glycol as a triblock polymer (i.e., PDLLA-PEG-PDLLA).Paclitaxel release from this polymer is shown in FIG. 41.

Example 33 Analysis of Drug Release

[0559] A known weight of a polymer (typically a 2.5 mg pellet) is addedto a 15 ml test tube containing 14 ml of a buffer containing 10 mmNa₂HPO₄—NaH₂PO₄, 0.145 m NaCl and 0.4 g/l bovine serum albumin. Thetubes are capped and tumbled at 37° C. At specific times all the 14 mlof the liquid buffer are removed and replaced with fresh liquid buffer.

[0560] The liquid buffer is added to 1 milliliter of methylene chlorideand shaken for 1 minute to extract all the paclitaxel into the methylenechloride. The aqueous phase is then removed and the methylene chloridephase is dried under nitrogen. The residue is then dissolved in 60%acetonitrile: 40% water and the solution is injected on to a HPLC systemusing the following conditions: C8 column (Beckman Instruments USA),mobile phase of 58%:5%:37% acetonitrile: methanol: water at a flow rateof 1 minute per minute.

[0561] For paclitaxel the collected buffer is then analyzed at 232 nm.For MTX the collected buffer is applied directly to the HPLC column withno need for extraction in methylene chloride MTX is analyzed at 302 nm.For Vanadium containing compounds the liquid buffer is analyzed directlyusing a UV/VIS spectrometer in the 200 to 300 nm range.

Example 34 Manufacture of Polymeric Compositions Contacting PCL andMePEG

[0562] A. Paclitaxel Release from PCL

[0563] Polycaprolactone containing various concentrations of paclitaxelwas prepared as described in Example 10. The release of paclitaxel overtime was measured by HPLC essentially as described above. Results areshown in FIG. 42.

[0564] B. Effect of MePEG on Paclitaxel Release

[0565] MePEG at various concentrations was formulated into PCL pastecontaining 20% paclitaxel, utilizing the methods described in Example10. The release of paclitaxel over time was measured by HPLC essentiallyas described above. Results of this study are shown in FIG. 43.

[0566] C. Effect of MePEG on the Melting Point of PCL

[0567] MePEG at various concentrations (formulated into PCL pastecontaining 20% paclitaxel) was analyzed for melting point using DSCanalysis at a heating rate of 2.5° C. per minute. Results are shown inFIGS. 44A (melting point vs. % MePEG) and 44B (percent increase in timeto solidify vs. % MePEG).

[0568] D. Tensile Strength of MePEG Containing PCL

[0569] PCL containing MePEG at various concentrations was tested fortensile strength and time to fail by a CT-40 Mechanical Strength Tester.Results are shown in FIG. 45.

[0570] E. Effect of γ-irradiation or the Release of Paclitaxel

[0571] PCL.MePEG (80:20) paste loaded with 20% paclitaxel was irradiatedand analyzed for paclitaxel release over time. Results are set forth inFIG. 46

[0572] In summary, based on the above experiments it can be concludedthat the addition of MePEG makes the polymer less brittle and more waxlike, reduces the melting point and increases the solidification time ofthe polymer. All these factors improve the application properties of thepaste. At low concentrations (20%) MePEG has no effect on the release ofpaclitaxel from PCL. Gamma-irradiation appears to have little effect onpaclitaxel release.

Example 35 Methotrexate-Loaded Paste

[0573] A Manufacture of Methotrexate-Loaded Paste

[0574] Methotrexate (“MTX”; Sigma Chemical Co.) is ground in a pestleand mortar to reduce the particle size to below 5 microns. It is thenmixed as a dry powder with polycaprolactone (molecular wt 18000Birmingham Polymers, AL USA). The mixture is heated to 65° C. for 5minutes and the molten polymer/methotrexate mixture is stirred into asmooth paste for 5 minutes. The molten paste is then taken into a 1 mLsyringe, and extruded as desired.

[0575] B. Results

[0576] Results are shown in FIGS. 47A-E. Briefly, FIG. 47A shows MTXrelease from PCL discs containing 20% MePEG and various concentrationsof MTX. FIG. 47B shows a similar experiment for paste which does notcontain MePEG. FIGS. 47C, D, and E show the amount of MTX remaining inthe disk.

[0577] As can be seen by the above results, substantial amounts of MTXcan be released from the polymer when high MePEG concentrations areutilized.

Example 36 Manufacture of Microspheres Containing Methotrexate

[0578] A. Microspheres With MTX Alone

[0579] Methotrexate (Sigma) was ground in a pestle and mortar to reducethe particle size to below 5 microns. One hundred milliliters of a2.5R/O PVA (w/v) (Aldrich or Sigma) in water was stirred for 15 minuteswith 500 mg of unground MTX at 25° C. to saturate the solution with MTX.This solution was then centrifuged at 2000 rpm to remove undissolved MTXand the supernatant used in the manufacture of microspheres.

[0580] Briefly, 10 ml of a 5% w/v solution of poly(DL) lactic acid(molecular weight 500,000; Polysciences), Polylactic:glycolic acid(50:50 IV 0.78 polysciences) or polycaprolactone (molecular weight18,000, BPI) containing 10:90 w/w MTX(ground):POLYMER were slowlydripped into 100 nL of the MTX saturated 2.5% w/v solution of PVA(Aldrich or Sigma) with stirring at 600 rpm. The mixture was stirred at25° C. for 2 hours and the resulting microspheres were washed and dried.

[0581] Using this method MTX loaded microspheres can be reproduciblymanufactured in the 30 to 160 micron size range.

[0582]FIG. 60 depicts the results for 10% methotrexate-loadedmicrospheres made from PLA:GA (50:50); Inherent Viscosity “IV”=0.78.

[0583] B. Microspheres With MTX and Hyaluronic Acid

[0584] MTX loaded microspheres can be made using hyaluronic acid (“HA”)as the carrier by a water in oil emulsion manufacture method,essentially as described below. Briefly, 50 ml of Parafin oil (lightoil; Fisher Scientific) is warmed to 60° C. with stirring at 200 rpm. A5 mL solution of sodium hyaluronate (20/mL); source=rooster comb; Sigma)in water containing various amounts MTX is added dropwise into theParafin oil. The mixture is stirred at 200 rpm for 5 hours. centrifugedat 500×g for 5 minutes. The resulting microspheres are washed in hexanefour times, and allowed to dry.

Example 37 Manufacture of Polymeric Compositions Containing VanadiumCompounds

[0585] A. Polymeric Paste Containing Vanadyl Sulfate

[0586] Vanadyl Sulfate (Fisher Scientific) is first round in a pestleand mortar to reduce the particle size, then dispersed into melted PCLas described above for MTX. It is then taken up into a syringe tosolidify and is ready for use.

[0587] Drug release was determined essentially as described above inExample 33, except that a 65 mg pellet of a 10% w/w VOSO₄:PCL wassuspended in 10 ml of water and the supernatant analyzed for releasedVanadyl Sulphate using UV/Vis absorbance spectroscopy of the peak in the200 to 300 nm range.

[0588] Results are shown in FIG. 61. Briefly, from a polymericcomposition containing 10% VOSO₄, 1 mg Of VOSO₄ was released in 6 hours,3 mg after 2 days and 5 mg by day 6.

[0589] B. Polymeric Microspheres Containing Vanadyl Sulfate

[0590] Vanadyl sulfate was incorporated into microspheres of polylacticacid or hyaluronic acid essentially as described in Example 36B. Resultsare shown in FIG. 62.

[0591] C. Polymeric Paste Containing Organic Vanadate

[0592] Organic vanadate is loaded into a PCL paste essentially asdescribed above in Example 35. Vanadate release from the microsphereswas determined as described above and in Example 33. Results are shownin FIGS. 63A and 63B.

[0593] D. Organic Vanadate Containing Microspheres

[0594] Organic vanadate may also be loaded into microspheres essentiallyas described in Example 36A. Such microspheres are shown in FIG. 64 forpoly D,L lactic acid (M.W. 500,000, Polysciences).

Example 38 Polymer Compositions with Increased Concentrations ofPaclitaxel

[0595] PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers withhydrophobic (PDLLA) and hydrophilic (PEG or MePEG) regions. Atappropriate molecular weights and chemical composition, they may formtiny aggregates of hydrophobic PDLLA core and hydrophilic MePEG shell.Paclitaxel can be loaded into the hydrophobic core, thereby providingpaclitaxel with an increased “solubility”.

[0596] A. Materials

[0597] D,L-lactide was purchased from Aldrich, Stannous octoate, poly(ethylene glycol) (mol. wt. 8,000), MePEG (mol. wt. 2,000 and 5,000)were from Sigma. MePEG (mol. wt. 750) was from Union Carbide. Thecopolymers were synthesized by a ring opening polymerization procedureusing stannous octoate as a catalyst (Deng et al, J. Polym. Sci., Polym.Lett. 28:411-416, 1990; Cohn et al, J. Biomed, Mater. Res. 22: 993-1009,1988).

[0598] For synthesizing PDLLA-MePEG, a mixture ofDL-lactide/MePEG/stannous octoate was added to a 10 milliliter glassampoule. The ampoule was connected to a vacuum and sealed with flame.Polymerization was accomplished by incubating the ampoule in a 150° C.oil bath for 3 hours. For synthesizing PDLLA-PEG-PDLLA, a mixture ofD,L-lactide/PEG/stannous octoate was transferred into a glass flask,sealed with a rubber stopper, and heated for 3 hours in a 150° C. oven.The starting compositions of the copolymers are given in Tables V andVI. In all the cases, the amount of stannous octoate was 0.5% -0.7%.

[0599] B. Methods

[0600] The polymers were dissolved in acetonitrile and centrifuged at10,000 g for 5 minutes to discard any non-dissolvable impurities.Paclitaxel acetonitrile solution was then added to each polymer solutionto give a solution with paclitaxel (paclitaxel-polymer) of 10%-wt. Thesolvent acetonitrile was then removed to obtain a clearpaclitaxel/PDLLA-MePEG matrix, under a stream of nitrogen and 60° C.warming. Distilled water, 0 9% NaCl saline, or 5% dextrose was added atfour times weight of the matrix. The matrix was finally “dissolved” withthe help of vortex mixing and periodic warming at 60° C. Clear solutionswere obtained in all the cases. The particle sizes were all below 50 nmas determined by a submicron particle sizer, NICOMP Model 270 Theformulations are given in Table V TABLE V Formulations ofPaclitaxel/PDLLA-MePEG* Paclitaxel Loading (final PDLLA-MePEG DissolvingMedia paclitaxel concentrate) 2000/50/50 water 10% (20 mg/ml) 2000/40/60water 10% (20 mg/ml) 2000/50/50 0.9% saline  5% (10 mg/ml) 2000/50/500.9% saline 10% (20 mg/ml) 2000/50/50 5% dextrose 10% (10 mg/ml)2000/50/50 5% dextrose 10% (20 mg/ml)

[0601] In the case of PDLLA-PEG-PDLLA since the copolymers cannotdissolve in water, paclitaxel and the polymer were co-dissolved inacetone. Water or a mixture of water/acetone was gradually added to thispaclitaxel polymer solution to induce the formation ofpaclitaxel/polymer spheres. TABLE VI Composition of PDLLA-PEG-PDLLACopolymer Name Wt. of PEG (g) Wt. of DL-lactide (g) PDLLA-PEG-PDLLA 1 990/10 PDLLA-PEG-PDLLA 2 8 80/20 PDLLA-PEG-PDLLA 3 7 70/30PDLLA-PEG-PDLLA 4 6 60/40 PDLLA-PEG-PDLLA 14 6 30-/70

[0602] C. Results

[0603] Many of the PDLLA-MePEG compositions form clear solutions inwater, 0.9% saline, or 5% dextrose, indicating the formation of tinyaggregates in the range of nanometers. Paclitaxel was loaded intoPDLLA-MePEG nanoparticles successfully. For example, at % loading (thisrepresents 10 mg paclitaxel in 1 ml paclitaxel/PDLLA-MePEG/aqueoussystem), a clear solution was obtained from 2000-50/50 and 2000-40/60.The particle size was about 20 nm.

Example 39 Insertion of Control and Paclitaxel Coated Stents intoMicroswine

[0604] As discussed above, various tubes within the body can be occludedby disease processes. One method for treating such occlusion is toinsert an endoluminal stent within the tube in order to relieve theobstruction. Unfortunately, the stents themselves are often overgrown byepithelial cells, thus limiting the duration and effectiveness of thetreatment. As described in more detail below, stainless steel stentswere coated with paclitaxel-loaded EVA polymer and placed into thebiliary duct of microswine in order to assess prevention of benignepithelial overgrowth.

[0605] A. Materials and Methods

[0606] Yucatan microswine were placed under general anesthetic and a 5cm transverse upper abdominal incision performed. The gallbladder wasgrasped and sewn to the anterior abdominal wall and a tiny incision wasmade in the gallbladder fundus. A 5F catheter was inserted into thegallbladder and radiopaque contrast injected to outline the biliarytree. A hydrophilic guidewire was advanced through the cystic duct intothe common bile duct, and over this a 7F (purpose—built, reusable)delivery catheter containing a stainless steel (5 mm diameter×4.2 cmlong) was advanced and deployed in the common bile duct. The deliverycatheter was withdrawn into the gallbladder and a repeat cholangiogramperformed. The gallbladder incision was closed, a radiopaque staple wasfixed at the incision site, and then the abdominal incision was closed.The swine was randomized into groups of receiving uncoated stents,polymer coated stents, and paclitaxel-loaded (33%) polymer coated stentsTantalum (strecker) stainless steel stents and stainless steelWallstents were used for each of these studies. Swines from each groupwere sacrificed at 14, 28, 56, and 112 days post-stent insertion byinjecting Euthanyl. After sacrifice, the gallbladder was cannulatedpercutaneously under X-ray by puncturing at the staple and radiopaquecontrast injected to outline the biliary tree. X-rays were taken andanalyzed for narrowing at or adjacent to the stent. The liver andbiliary tree were removed en bloc. The portion of bile duct containingthe stent was sectioned transversely at 1 cm intervals and thehistologic sections were used to assess the degree of overgrowth of thestent. The liver was also examined histologically for signs of chronicobstruction or inflammation.

[0607] B. Results

[0608] Control, uncoated stainless steel stents were inserted intomicroswine as described above, and sacrificed at various times. At twoweeks, the bile mucosa appeared normal in 2 of the sacrificed pigs,while one presented a small non-obstructive bile concretion within thebiliary lumen, and a slight indentation in the bile duct mucosa at thesite of the tines. At 4 weeks, of the 3 pigs which were sacrificed asmall bile concretion was present on the distal stent, as well asmucosal indentations of the stent tine within the bile duct mucosa. At 8weeks, the bile duct mucosa at the site of stent insertion in thesacrificed pigs partially overgrew the stent tines in a crescenticmanner over approximately 25-30% of the radius of the stent (FIGS. 65A,66A, and 66B). In addition, one pig contained thick bile containinginflammatory cells within the lumen. At 16 weeks, pigs which weresacrificed presented a stent which was completely overgrown distally byfibrous tissue, and no evidence of a lumen (FIG. 65B). Histologically,the tissue was uniformly fibrous. Surprisingly, the liver biopsy of allof the control treated swines were normal and there was no evidence ofobstructive changes.

[0609] In another group of microswine, stents coated with ethylene vinylacetate and 33% paclitaxel were inserted into the biliary duct. After an8 week exposure, one pig was sacrificed and showed a slight indentationof the bile duct mucosa at the site of the stent tines and no indicationof overgrowth (FIGS. 66C and 66D). The underlying mucosa was normalapart from some inflammatory cell infiltration. Non-obstructive bileconcretions were noted in the lumen of the stent. The liver biopsy wasnormal, with no evidence of obstructive changes.

[0610] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 1 9 amino acids amino acid single linear peptide N-terminal 1 Cys AspPro Gly Tyr Ile Gly Ser Arg 1 5

We claim:
 1. A composition, comprising: (a) a compound which disruptsmicrotubule function; and (b) a polymeric carrier, with the proviso thatsaid polymeric carrier is not a capsule.
 2. The composition according toclaim 1 wherein said composition is formed into microspheres having anaverage size of between 0.5 and 200 μm.
 3. The composition according toclaim 1 wherein said composition is formed into a film with a thicknessof between 100 μm and 2 mm.
 4. The composition according to claim 1wherein said composition is liquid above 45° C., and solid or semi-solidat 37° C.
 5. The composition according to claim 1 wherein said polymericcarrier is poly(ethylene-vinyl acetate) (40% crosslinked).
 6. Thecomposition according to claim 1 wherein said polymeric carrier iscopolymer of lactic acid and glycolic acid.
 7. The composition accordingto claim 1 wherein said polymeric carrier is poly (caprolactone).
 8. Thecomposition according to claim 1 wherein said polymeric carrier is poly(lactic acid).
 9. The composition according to claim 1 wherein saidpolymeric carrier is a copolymer of poly (lactic acid) and poly(caprolactone).
 10. The composition according to claim 1 wherein saidcompound which disrupts microtubule function is paclitaxel.
 11. Thecomposition according to claim 1 wherein said compound which disruptsmicrotubule function is selected from the group consisting ofestramustine, colchicine, methotrexate, curacin-A, epothilone,vinblastine and tBCEU.
 12. A composition, comprising: (a) ananti-angiogenic factor, and (b) hyaluronic acid.
 13. The composition ofclaim 10 wherein said anti-angiogenic factor is selected from the groupconsisting of paclitaxel, suramin, methotrexate and lighter d grouptransition metals.
 14. A composition comprising: (a) a lighter d grouptransition metal which inhibits the formation of new blood vessels; and(b) a polymeric carrier.
 15. The composition according to claim 14wherein said lighter d group is selected from the group consisting ofspecies of vanadium, molybdenum, tungsten, titanium, niobium andtantalum.
 16. A method for embolizing a blood vessel, comprisingdelivering into said vessel a therapeutically effective amount ofcomposition according to any one of claims 1-15, such that said bloodvessel is effectively occluded.
 17. The method according to claim 16wherein said blood vessel nourishes a tumor.
 18. A stent, comprising agenerally tubular structure, the surface of which is coated with acomposition comprising an anti-angiogenic factor and a polymericcarrier.
 19. A method for expanding the lumen of a body passageway,comprising inserting a stent into the passageway, the stent having agenerally tubular structure, the surface of said structure being coatedwith a composition comprising an anti-angiogenic factor and a polymericcarrier, such that said passageway is expanded.
 20. A method foreliminating vascular obstructions, comprising inserting a vascular stentinto a vascular passageway, the stent having a generally tubularstructure, the surface of said structure being coated with a compositioncomprising an anti-angiogenic factor and a polymeric carrier, such thatsaid vascular obstruction is eliminated.
 21. A method for eliminatingbiliary obstructions, comprising inserting a biliary stent into abiliary passageway, the stent having a generally tubular structure, thesurface of said structure being coated with a composition comprising ananti-angiogenic factor and a polymeric carrier, such that said biliaryobstruction is eliminated.
 22. A method for eliminating urethralobstructions, comprising inserting a urethral stent into a urethra, thestent having a generally tubular structure, the surface of saidstructure being coated with a composition comprising an anti-angiogenicfactor and a polymeric carrier, such that said urethral obstruction iseliminated.
 23. A method for eliminating esophageal obstructions,comprising inserting an esophageal stent into an esophagus, the stenthaving a generally tubular structure, the surface of said structurebeing coated with a composition comprising an anti-angiogenic factor anda polymeric carrier, such that said esophageal obstruction iseliminated.
 24. A method for eliminating tracheal/bronchialobstructions, comprising inserting a tracheal/bronchial stent into thetrachea or bronchi, the stent having a generally tubular structure, thesurface of which is coated with a composition comprising ananti-angiogenic factor and a polymeric carrier, such that saidtracheal/bronchial obstruction is eliminated.
 25. A method for treatinga tumor excision site, comprising administering a composition comprisingan anti-angiogenic factor and a polymeric carrier to the resectionmargin of a tumor subsequent to excision, such that the local recurrenceof cancer and the formation of new blood vessels at said site isinhibited.
 26. The method according to claim 25 wherein said compositionis liquid above 45° C. and solid or semi-solid at 37° C.
 27. The methodaccording to claim 26 wherein the step of administering comprisesspraying microspheres composed of said composition into the resectionmargin of the tumor.
 28. A method for treating cornealneovascularization, comprising administering to a patient atherapeutically effective amount of a composition comprising ananti-angiogenic factor and a polymeric carrier to the cornea, such thatthe formation of blood vessels is inhibited.
 29. A method for treatingcorneal neovascularization, comprising administering to a patient atherapeutically effective amount of paclitaxel to the cornea, such thatthe formation of blood vessels is inhibited.
 30. The method according toclaims 28 or 29 wherein said method further comprises the administrationof a topical corticosteroid.
 31. A method for inhibiting angiogenesis inpatients with non-tumorigenic, angiogenesis-dependent diseases,comprising administering to a patient a therapeutically effective amountof a composition comprising paclitaxel to a patient with anon-tumorigenic angiogenesis-dependent disease, such that the formationof new blood vessels is inhibited.
 32. A method for embolizing a bloodvessel in a non-tumorigenic, angiogenesis-dependent diseases, comprisingdelivering to said vessel a therapeutically effective amount of acomposition comprising paclitaxel, such that said blood vessel iseffectively occluded.
 33. A method for expanding the lumen of a bodypassageway, comprising inserting a stent into the passageway, the stenthaving a generally tubular structure, the surface of said structurebeing coated with a composition comprising paclitaxel, such that saidpassageway is expanded.
 34. A method for eliminating vascularobstructions, comprising inserting a vascular stent into a vascularpassageway, the stent having a generally tubular structure, the surfaceof said structure being coated with a composition comprising paclitaxel,such that said vascular obstruction is eliminated.
 35. A method foreliminating biliary obstructions, comprising inserting a biliary stentinto a biliary passageway, the stent having a generally tubularstructure, the surface of said structure being coated with a compositioncomprising paclitaxel, such that said biliary obstruction is eliminated.36. A method for eliminating urethral obstructions, comprising insertinga urethral stent into a urethra, the stent having a generally tubularstructure, the surface of said structure being coated with a compositioncomprising paclitaxel, such that said urethral obstruction iseliminated.
 37. A method for eliminating esophageal obstructions,comprising inserting an esophageal stent into an esophagus, the stenthaving a generally tubular structure, the surface of said structurebeing coated with a composition comprising paclitaxel, such that saidesophageal obstruction is eliminated.
 38. A method for eliminatingtracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi, the stent having agenerally tubular structure, the surface of said structure being coatedwith a composition comprising paclitaxel, such that saidtracheal/bronchial obstruction is eliminated.
 39. A method for treatinga tumor excision site, comprising administering to a patient acomposition comprising paclitaxel to the resection margin of a tumorsubsequent to excision, such that the local recurrence of cancer and theformation of new blood vessels at said site is inhibited.
 40. A methodfor treating neovascular disease of the eye, comprising administering toa patient a therapeutically effective amount of a compound whichdisrupts microtubule function to the cornea, such that the formation ofnew vessels is inhibited.
 41. The method according to claim 40 whereinsaid compound which disrupts microtubule function is paclitaxel.
 42. Themethod according to claim 40 wherein said compound which disruptsmicrotubule function is selected from the group consisting ofestramustine, colchicine, methotrexate, curacin-A, epothilone,vinblastine and tBCEU.
 43. The method according to claim 40 wherein saidneovascular disease is selected from the group consisting of cornealneovascularization and macular degeneration.
 44. A method for treatinginflammatory arthritis, comprising administering to a patient atherapeutically effective amount of a composition comprising ananti-angiogenic factor and a polymeric carrier to a joint, such that theformation of blood vessels is inhibited, with the proviso that saidanti-angiogenic factor is not methotrexate.
 45. The method according toclaim 44 wherein said anti-angiogenic factor is a compound whichdisrupts microtubule function.
 46. The method according to claim 44wherein said compound which disrupts microtubule function is selectedfrom the group consisting of estramustine, colchicine, curacin-A,epothilone, vinblastine and tBCEU.
 47. The method according to claim 44wherein said compound which disrupts microtubule function is paclitaxel.48. A method for inflammatory arthritis, comprising administering to apatient a therapeutically effective amount of an anti-angiogenic factorto a joint, such that the formation of blood vessels is inhibited, withthe proviso that said anti-angiogenic factor is not methotrexate. 49.The method according to claim 48 wherein said anti-angiogenic factor isa compound which disrupts microtubule function.
 50. The method accordingto claim 48 wherein said compound which disrupts microtubule function isselected from the group consisting of estramustine. colchicine,curacin-A, epothilone, vinblastine and tBCEU.
 51. The method accordingto claim 48 wherein said compound which disrupts microtubule function ispaclitaxel.
 52. A composition comprising a polymeric carrier adapted tocontain and release a hydrophobic compound, said carrier containing ahydrophobic compound in combination with a carbohydrate, protein orpolypeptide.
 53. The composition of claim 52 wherein said polymericcarrier is poly(ethylene-vinyl acetate) (40% crosslinked).
 54. Thecomposition of claim 52 wherein said polymeric carrier is copolymer oflactic acid and glycolic acid.
 55. The composition of claim 52 whereinsaid polymeric carrier is poly (caprolactone).
 56. The composition ofclaim 52 wherein said polymeric carrier is poly (lactic acid).
 57. Thecomposition according to claim 52 wherein said hydrophobic compound is acompound which disrupts microtubule function.
 58. The compositionaccording to claim 57 wherein said compound which disrupts microtubulefunction is paclitaxel.
 59. The composition according to claim 57wherein said compound which disrupts microtubule function is selectedfrom the group consisting of estramustine, colchicine, methotrexate,curacin-A, epothilone, vinblastine and tBCEU.
 60. A pharmaceuticalproduct, comprising: (a) a compound which disrupts microtubule function,in a container; and (b) a notice associated with said container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by saidagency of said paclitaxel, for human or veterinary administration totreat non-tumorigenic angiogenesis-dependent diseases.
 61. Thepharmaceutical product according to claim 60 wherein said compound whichdisrupts microtubule function is paclitaxel.